Zap-70 expression as a marker for chronic lymphocytic leukemia / small lymphocytic lymphoma (cll/sll)

ABSTRACT

It has been surprisingly found that ZAP-70 expression, both at the protein and mRNA levels, is indicative of clinical subgroups of CLL/SLL patients. In particular, high ZAP-70 expression is indicative of Ig-unmutated CLL/SLL. Methods are provided for discriminating between clinical subgroups of CLL/SLL, by determining whether subjects overexpress ZAP-70 mRNA mRNA or protein.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 11/955,322,filed on Dec. 12, 2007, which is a divisional of U.S. patent applicationSer. No. 10/309,548, filed on Dec. 3, 2002, issued as U.S. Pat. No.7,329,502 on Feb. 12, 2008, which claims the benefit of U.S. ProvisionalApplication No. 60/375,966, filed on Apr. 25, 2002, now expired, all ofwhich are herein incorporated by reference in their entirety.

FIELD

This disclosure relates to methods of diagnosis and detection ofcancers, and more particularly to distinguishing types of CLL/SLL basedon the level of ZAP-70 protein or nucleic acid in a biological sample

BACKGROUND

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is amalignancy of B-lymphocytes in the blood, bone marrow, and lymph nodeswith a characteristic immunophenotype. The recent WHO classificationdiscusses CLL/SLL as an entity but notes that the term SLL is restrictedto cases with the tissue morphology and immunophenotype of CLL, butwhich are non-leukemic (WHO Classification of Tumours. Tumours ofHaemotopoietic and Lymphoid Tissue. Edited by Jaffe, Harris, Stein,Vardiman. IARC Press 2001). The clinical course of CLL is quite varied.While some patients have a chronic lymphocytosis without any need fortherapeutic interventions, other patients may die rapidly despiteaggressive treatment. The classic staging systems provide only limitedprognostic information in newly diagnosed patients.

Recently, the presence or absence of somatic mutations in theimmunoglobulin (Ig) variable region genes has been shown to distinguishbetween two disease subsets conferring important prognostic information.A median survival of 95 months was found in patients with unmutated Iggenes versus 293 months in patients with mutated Ig genes (Hamblin,Blood 94(6):1848-1854, 1999). Unfortunately, the ability to sequence Iggenes is not available in most clinical laboratories.

In addition to mutated Ig genes, several other potential diagnostic orprognostic markers have been identified for CLL, as well as for othersmall B-cell lymphomas. By way of example, these include CD10, CD20,CD21, CD23 (including serum CD23), CD38, CD69, CD43, FMC-7, and BCL-6.The research and medical communities are actively searching for goodprognostic markers, but as yet no definitive markers have beenidentified.

SUMMARY

This disclosure provides a method of detecting a biological conditionassociated with ZAP-70 overexpression in a subject. Also provided hereinare methods to determine whether a subject has ZAP-70 nucleic acid orZAP-70 protein overexpression. It is shown herein that the biologicalcondition associated with ZAP-70 overexpression is Ig-unmutated CLL.

The disclosure also provides a method of modifying a level of expressionof a ZAP-70 protein in a subject in order to reduce, ameliorate, orcontrol CLL. Examples of these methods include expressing in the subjecta recombinant genetic construct including a promoter operably linked toa nucleic acid molecule where expression of the nucleic acid moleculechanges expression of the ZAP-70 protein. In one embodiment, the nucleicacid molecule includes at least 15 consecutive nucleotides of thenucleotide sequence shown in SEQ ID NO: 1. In another embodiment, thenucleic acid sequence includes a sequence at least 85% identical to SEQID NO: 1.

Also provided herein are kits for determining whether or not a subjecthas a biological condition associated with ZAP-70 overexpression. In oneembodiment, the kit is an in vitro assay kit. These kits can be used todetect an overabundance of ZAP-70 protein or nucleic acid in a sample oftissue and/or body fluids from the subject. For example, the kits caninclude a container with an antibody specific for ZAP-70 protein andinstructions for using the kit. The instructions can indicate the stepsfor performing a method to detect the presence of ZAP-70 protein ornucleic acid in the sample as well as how to analyze data generated bythe method. In one embodiment, the instructions indicate thatoverabundance of ZAP-70 protein in the sample indicates that theindividual has or is predisposed to a biological condition.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and B is a series of schematic drawings showing the statisticalmethodology for the creation and validation of an Ig-mutational statuspredictor in CLL. FIG. 1A shows the performance of the predictor using across-validation strategy. FIG. 1B shows the performance of theIg-mutational subtype predictor in a test set of six unmutated (*) andfour mutated CLL (Δ) samples.

FIG. 2 is a graph showing the predictive value of ZAP-70 mRNA andprotein expression as surrogate markers of IgVH mutation status in CLL.ZAP-70 mRNA expression levels, as determined by DNA microarray analysis,predicted Ig-mutation status correctly in 95% of patients. ZAP-70protein expression as determined by immunohistochemistry predictedIg-mutation status correctly in 86% of patients.

FIGS. 3A and B is a series of graphs showing the impact of ZAP-70 mRNAand Ig-mutation status on the clinical course of CLL. Rate of diseaseprogression is shown, as assessed by the treatment-free time intervalmeasured in months from diagnosis for IgVH mutation status (FIG. 3A) andZAP-70 mRNA expression (FIG. 3B).

FIG. 4 is a graph showing that quantitative RT-PCR could serve as aclinical test of ZAP-70 mRNA expression. Real time quantitative RT-PCRwas performed in 9 CLL samples representing the ZAP-70 mRNA expressionspectrum defined by the DNA microarray analysis. ZAP-70 expression isshown relative to the expression of beta-2-microglobulin in the samesample. The Pearson coefficient for correlation between the two methodswas r=0.941.

FIGS. 5A and B is a series of digital images showing that ZAP-70 proteinexpression can distinguish CLL subtypes and could serve as a clinicaltest. In FIG. 5A, ZAP-70 protein expression was assessed by Westernblotting in whole cell lysates of normal peripheral blood mononuclearcells (PBMC), or CD19+ purified leukemic cells from blood of patientswith Ig-unmutated and Ig-mutated CLL. The data are representative ofWestern blot analysis of 20 patient samples analyzed. Equal loading isdemonstrated by probing for beta-tubulin. In FIG. 5B, ZAP-70 can bedetected by immunohistochemistry in clinical samples. PBMC (upper half)were embedded in a fibrin clot, fixed and processed by standardtechniques. PBMC and routine bone marrow trephine biopsies (lower half)were stained with CD20 demonstrating involvement by B cell CLL (B-CLL),and CD3, which stains interspersed T-cells. ZAP-70 was positive in Tcells and Ig-unmutated CLL cells.

SEQUENCE LISTING (INFORMAL)

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. In the accompanying sequence listing:

SEQ ID NO: 1 shows a cDNA encoding ZAP-70.SEQ ID NO: 2 shows the amino acid sequence of ZAP-70.SEQ ID NO: 3 shows the forward or upstream ZAP-70 oligonucleotide primer(5′ TCTCCAAAGCACTGGGTG 3′).SEQ ID NO: 4 shows the reverse or downstream ZAP-70 oligonucleotideprimer (5′ AGCTGTGTGTGGAGACAACCAAG 3′).SEQ ID NO: 5 shows the forward or upstream VH1 and VH7 primer (5′-CCATGG ACT GGA CCT GGA-3′).SEQ ID NO: 6 shows the forward or upstream VH2 primer (5′-ATG GAC ATACTT TGT TCC AC-3′).SEQ ID NO: 7 shows the forward or upstream VH3 primer (5′-CCA TGG AGTTTG GGC TGA GC-3′).SEQ ID NO: 8 shows the forward or upstream VH4 primer (5′-ATG AAA CACCTG TGG TTC TT-3′).SEQ ID NO: 9 shows the forward or upstream VH5 primer (5′-ATG GGG TCAACC GCC ATC CT-3′).SEQ ID NO: 10 shows the forward or upstream VH6 primer (5′-ATG TCT GTCTCC TTC CTC AT-3′).SEQ ID NO: 11 shows a 3′ oligonucleotide complementary to the JHconsensus sequence (5′-ACC TGA GGA GAC GGT GAC C-3′) as a reverse ordownstream primer.SEQ ID NO: 12 shows the constant region of the IgM locus (5′-AGG AGA AAGTGA TGG AGT CG-3′) as a reverse or downstream primer.SEQ ID NO: 13 shows the forward ZAP-70 primer.SEQ ID NO: 14 shows the reverse ZAP-70 primer.SEQ ID NO: 15 shows the ZAP-70 FAM™-probe.SEQ ID NO: 16 shows the framework region (FR)1-VH1 forward primer,SEQ ID NO: 17 shows the framework region (FR)1-VH2 forward primer.SEQ ID NO: 18 shows the framework region (FR)1-VH3 forward primer.SEQ ID NO: 19 shows the framework region (FR)1-VH4 forward primer.SEQ ID NO: 20 shows the framework region (FR)1-VH5 forward primer.SEQ ID NO: 21 shows the framework region (FR)1-VH6 forward primer.

DETAILED DESCRIPTION I. Abbreviations

BCR B cell receptorB-CLL B cell CLLCLL chronic lymphocytic leukemiaDLBCL diffuse large B cell lymphomaFGFR fibroblast growth factor receptorH heavyIgV Ig variable region

M-CLL IgV-mutated CLL

PBMC peripheral blood mononuclear cellsPKC protein kinase CRT-PCR reverse transcription polymerase chain reactionSLL small lymphocytic lymphomaTCR T cell antigen receptor

UM-CLL IgV-unmutated CLL II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of theinvention, the following explanations of specific terms are provided:

Antisense, Sense, and Antigene: Double-stranded DNA (dsDNA) has twostrands, a 5′->3′ strand, referred to as the plus strand, and a 3′->5′strand (the reverse complement), referred to as the minus strand.Because RNA polymerase adds nucleic acids in a 5′->3′ direction, theminus strand of the DNA serves as the template for the RNA duringtranscription. Thus, the RNA formed will have a sequence complementaryto the minus strand and identical to the plus strand (except that U issubstituted for T).

Antisense molecules are molecules that are specifically hybridizable orspecifically complementary to either RNA or the plus strand of DNA.Sense molecules are molecules that are specifically hybridizable orspecifically complementary to the minus strand of DNA. Antigenemolecules are either antisense or sense molecules directed to a dsDNAtarget.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and transcriptional regulatory sequences. cDNA mayalso contain untranslated regions (UTRs) that are responsible fortranslational control in the corresponding RNA molecule. cDNA is usuallysynthesized in the laboratory by reverse transcription from messengerRNA extracted from cells.

DNA (deoxyribonucleic acid): DNA is a long chain polymer which comprisesthe genetic material of most living organisms (some viruses have genescomprising ribonucleic acid (RNA)). The repeating units in DNA polymersare four different nucleotides, each of which comprises one of the fourbases, adenine (A), guanine (G), cytosine (C), and thymine (T) bound toa deoxyribose sugar to which a phosphate group is attached. Triplets ofnucleotides (referred to as codons) code for each amino acid in apolypeptide, or for a stop signal. The term codon is also used for thecorresponding (and complementary) sequences of three nucleotides in themRNA into which the DNA sequence is transcribed.

Unless otherwise specified, any reference to a DNA molecule is intendedto include the reverse complement of that DNA molecule. Except wheresingle-strandedness is required by the text herein, DNA molecules,though written to depict only a single strand, encompass both strands ofa double-stranded DNA molecule. Thus, a reference to the nucleic acidmolecule that encodes a specific protein, or a fragment thereof,encompasses both the sense strand and its reverse complement. Thus, forinstance, it is appropriate to generate probes or primers from thereverse complement sequence of the disclosed nucleic acid molecules.

Hybridization: Oligonucleotides and their analogs hybridize by hydrogenbonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary bases. Generally, nucleic acidconsists of nitrogenous bases that are either pyrimidines (cytosine (C),uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)).These nitrogenous bases form hydrogen bonds between a pyrimidine and apurine, and the bonding of the pyrimidine to the purine is referred toas “base pairing.” More specifically, A will hydrogen bond to T or U,and G will bond to C. “Complementary” refers to the base pairing thatoccurs between to distinct nucleic acid sequences or two distinctregions of the same nucleic acid sequence.

“Specifically hybridizable” and “specifically complementary” are termsthat indicate a sufficient degree of complementarity such that stableand specific binding occurs between the oligonucleotide (or its analog)and the DNA or RNA target. The oligonucleotide or oligonucleotide analogneed not be 100% complementary to its target sequence to be specificallyhybridizable. An oligonucleotide or analog is specifically hybridizablewhen binding of the oligonucleotide or analog to the target DNA or RNAmolecule interferes with the normal function of the target DNA or RNA,and there is a sufficient degree of complementarity to avoidnon-specific binding of the oligonucleotide or analog to non-targetsequences under conditions where specific binding is desired, forexample under physiological conditions in the case of in vivo assays orsystems. Such binding is referred to as specific hybridization.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing nucleic acidsequences. Generally, the temperature of hybridization and the ionicstrength (especially the Na⁺ concentration) of the hybridization bufferwill determine the stringency of hybridization, though waste times alsoinfluence stringency. Calculations regarding hybridization conditionsrequired for attaining particular degrees of stringency are discussed bySambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed.,vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989, chapters 9 and 11, herein incorporated by reference.

For present purposes, “stringent conditions” encompass conditions underwhich hybridization will only occur if there is less than 25% mismatchbetween the hybridization molecule and the target sequence. “Stringentconditions” may be broken down into particular levels of stringency formore precise definition. Thus, as used herein, “moderate stringency”conditions are those under which molecules with more than 25% sequencemismatch will not hybridize; conditions of “medium stringency” are thoseunder which molecules with more than 15% mismatch will not hybridize,and conditions of “high stringency” are those under which sequences withmore than 10% mismatch will not hybridize. Conditions of “very highstringency” are those under which sequences with more than 6% mismatchwill not hybridize.

In vitro amplification: Techniques that increases the number of copiesof a nucleic acid molecule in a sample or specimen. An example ofamplification is the polymerase chain reaction, in which a biologicalsample collected from a subject is contacted with a pair ofoligonucleotide primers, under conditions that allow for thehybridization of the primers to nucleic acid template in the sample. Theprimers are extended under suitable conditions, dissociated from thetemplate, and then re-annealed, extended, and dissociated to amplify thenumber of copies of the nucleic acid. The product of in vitroamplification may be characterized by electrophoresis, restrictionendonuclease cleavage patterns, oligonucleotide hybridization orligation, and/or nucleic acid sequencing, using standard techniques.Other examples of in vitro amplification techniques include stranddisplacement amplification (see U.S. Pat. No. 5,744,311);transcription-free isothermal amplification (see U.S. Pat. No.6,033,881); repair chain reaction amplification (see WO 90/01069);ligase chain reaction amplification (see EP-A-320 308); gap fillingligase chain reaction amplification (see U.S. Pat. No. 5,427,930);coupled ligase detection and PCR (see U.S. Pat. No. 6,027,889); andNASBA™ RNA transcription-free amplification (see U.S. Pat. No.6,025,134).

Isolated: An “isolated” biological component (such as a nucleic acidmolecule, protein, cell, or organelle) has been substantially separatedor purified away from other biological components in the cell of theorganism in which the component naturally occurs, i.e., otherchromosomal and extra-chromosomal DNA and RNA, proteins, and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinantexpression in a host cell as well as chemically synthesized nucleicacids.

Nucleotide: “Nucleotide” includes, but is not limited to, a monomer thatincludes a base linked to a sugar, such as a pyrimidine, purine orsynthetic analogs thereof, or a base linked to an amino acid, as in apeptide nucleic acid (PNA). A nucleotide is one monomer in apolynucleotide. A nucleotide sequence refers to the sequence of bases ina polynucleotide.

Oligonucleotide: An oligonucleotide is a plurality of joined nucleotidesjoined by phosphodiester bonds, between about 6 and about 500nucleotides in length. An oligonucleotide analog refers to moieties thatfunction similarly to oligonucleotides but have non-naturally occurringportions. For example, oligonucleotide analogs can contain altered sugarmoieties or inter-sugar linkages, such as a phosphorothioateoligodeoxynucleotide. Functional analogs of naturally occurringpolynucleotides can bind to RNA or DNA, and include peptide nucleic acid(PNA) molecules.

Particular oligonucleotides and oligonucleotide analogs can includelinear sequences up to about 300 nucleotides in length, for example asequence (such as DNA or RNA) that is at least 6 bases, for example atleast 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or even 200 or morebases long, or from about 6 to about 50 bases, for example about 10-25bases, such as 12, 15, 20, or 25 bases.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Open reading frame: A series of nucleotide triplets (codons) coding foramino acids without any internal termination codons. These sequences areusually translatable into a peptide.

Peptide Nucleic Acid (PNA): An oligonucleotide analog with a backbonecomprised of monomers coupled by amide (peptide) bonds, such as aminoacid monomers joined by peptide bonds.

Polymorphism: Variant in a sequence of a gene. Polymorphisms can bethose variations (nucleotide sequence differences) that, while having adifferent nucleotide sequence, produce functionally equivalent geneproducts, such as those variations generally found between individuals,different ethnic groups, geographic locations. The term polymorphismalso encompasses variations that produce gene products with alteredfunction, i.e., variants in the gene sequence that lead to gene productsthat are not functionally equivalent. This term also encompassesvariations that produce no gene product, an inactive gene product, orincreased gene product. The term polymorphism may be usedinterchangeably with allele or mutation, unless context clearly dictatesotherwise.

Polymorphisms can be referred to, for instance, by the nucleotideposition at which the variation exists, by the change in amino acidsequence caused by the nucleotide variation, or by a change in someother characteristic of the nucleic acid molecule that is linked to thevariation (e.g., an alteration of a secondary structure such as astem-loop, or an alteration of the binding affinity of the nucleic acidfor associated molecules, such as polymerases, RNases, and so forth).

Probes and primers: Nucleic acid probes and primers can be readilyprepared based on the nucleic acid molecules provided as indicators ofdisease or disease progression. It is also appropriate to generateprobes and primers based on fragments or portions of these nucleic acidmolecules. Also appropriate are probes and primers specific for thereverse complement of these sequences, as well as probes and primers to5′ or 3′ regions.

A probe comprises an isolated nucleic acid attached to a detectablelabel or other reporter molecule. Typical labels include radioactiveisotopes, enzyme substrates, co-factors, ligands, chemiluminescent orfluorescent agents, haptens, and enzymes. Methods for labeling andguidance in the choice of labels appropriate for various purposes arediscussed, e.g., in Sambrook et al. (In Molecular Cloning: A LaboratoryManual, CSHL, New York, 1989) and Ausubel et al. (In Current Protocolsin Molecular Biology, John Wiley & Sons, New York, 1998).

Primers are short nucleic acid molecules, for instance DNAoligonucleotides 10 nucleotides or more in length. Longer DNAoligonucleotides may be about 15, 20, 25, 30 or 50 nucleotides or morein length. Primers can be annealed to a complementary target DNA strandby nucleic acid hybridization to form a hybrid between the primer andthe target DNA strand, and then the primer extended along the target DNAstrand by a DNA polymerase enzyme. Primer pairs can be used foramplification of a nucleic acid sequence, e.g., by the polymerase chainreaction (PCR) or other in vitro nucleic-acid amplification methodsknown in the art.

Methods for preparing and using nucleic acid probes and primers aredescribed, for example, in Sambrook et al. (In Molecular Cloning: ALaboratory Manual, CSHL, New York, 1989), Ausubel et al. (ed.) (InCurrent Protocols in Molecular Biology, John Wiley & Sons, New York,1998), and Innis et al. (PCR Protocols, A Guide to Methods andApplications, Academic Press, Inc., San Diego, Calif., 1990).Amplification primer pairs (for instance, for use with polymerase chainreaction amplification) can be derived from a known sequence such as theZAP-70 sequences described herein, for example, by using computerprograms intended for that purpose such as Primer (Version 0.5, 1991,Whitehead Institute for Biomedical Research, Cambridge, Mass.).

One of ordinary skill in the art will appreciate that the specificity ofa particular probe or primer increases with its length. Thus, forexample, a primer comprising 30 consecutive nucleotides of a ZAP-70protein-encoding nucleotide will anneal to a target sequence, such asanother homolog of the designated ZAP-70 protein, with a higherspecificity than a corresponding primer of only 15 nucleotides. Thus, inorder to obtain greater specificity, probes and primers can be selectedthat comprise at least 20, 23, 25, 30, 35, 40, 45, 50 or moreconsecutive nucleotides of a ZAP-70 protein-encoding nucleotidesequences.

Also provided are isolated nucleic acid molecules that comprisespecified lengths of the disclosed ZAP-70 nucleotide sequences. Suchmolecules may comprise at least 10, 15, 20, 23, 25, 30, 35, 40, 45 or 50or more (e.g., at least 100, 150, 200, 250, 300 and so forth)consecutive nucleotides of these sequences or more. These molecules maybe obtained from any region of the disclosed sequences (e.g., a ZAP-70nucleic acid may be apportioned into halves or quarters based onsequence length, and isolated nucleic acid molecules may be derived fromthe first or second halves of the molecules, or any of the fourquarters, etc.). A ZAP-70 cDNA or other encoding sequence also can bedivided into smaller regions, e.g. about eighths, sixteenths,twentieths, fiftieths, and so forth, with similar effect.

Another mode of division is to select the 5′ (upstream) and/or 3′(downstream) region associated with a ZAP-70 gene.

Protein: A biological molecule expressed by a gene or recombinant orsynthetic coding sequence and comprised of amino acids.

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purified proteinpreparation is one in which the protein referred to is more pure thanthe protein in its natural environment within a cell or within aproduction/reaction chamber (as appropriate).

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination can be accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques. A recombinantorganism or cell is one that comprises at least one recombinant nucleicacid molecule.

Sequence identity: The similarity between two nucleic acid sequences, ortwo amino acid sequences, is expressed in terms of the similaritybetween the sequences, otherwise referred to as sequence identity.Sequence identity is frequently measured in terms of percentage identity(or similarity or homology); the higher the percentage, the more similarthe two sequences are. Homologs or orthologs of human ZAP-70 protein,and the corresponding cDNA or gene sequence, will possess a relativelyhigh degree of sequence identity when aligned using standard methods.This homology will be more significant when the orthologous proteins orgenes or cDNAs are derived from species that are more closely related(e.g., human and chimpanzee sequences), compared to species moredistantly related (e.g., human and C. elegans sequences).

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman Adv. Appl. Math. 2: 482, 1981; Needleman & Wunsch J. Mol. Biol.48: 443, 1970; Pearson & Lipman Proc. Natl. Acad. Sci. USA 85: 2444,1988; Higgins & Sharp Gene, 73: 237-244, 1988; Higgins & Sharp CABIOS 5:151-153, 1989; Corpet et al. Nuc. Acids Res. 16, 10881-90, 1988; Huanget al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearsonet al. Meth. Mol. Bio. 24, 307-31, 1994. Altschul et al. (J. Mol. Biol.215:403-410, 1990), presents a detailed consideration of sequencealignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. J.Mol. Biol. 215:403-410, 1990) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.By way of example, for comparisons of amino acid sequences of greaterthan about 30 amino acids, the Blast 2 sequences function is employedusing the default BLOSUM62 matrix set to default parameters, (gapexistence cost of 11, and a per residue gap cost of 1). When aligningshort peptides (fewer than around 30 amino acids), the alignment isperformed using the Blast 2 sequences function, employing the PAM30matrix set to default parameters (open gap 9, extension gap 1penalties).

An alternative indication that two nucleic acid molecules are closelyrelated is that the two molecules hybridize to each other understringent conditions. Stringent conditions are sequence-dependent andare different under different environmental parameters. Generally,stringent conditions are selected to be about 5° C. to 20° C. lower thanthe thermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence remains hybridizedto a perfectly matched probe or complementary strand. Conditions fornucleic acid hybridization and calculation of stringencies can be foundin Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, NewYork, 1989) and Tijssen (Laboratory Techniques in Biochemistry andMolecular Biology—Hybridization with Nucleic Acid Probes Part I, Chapter2, Elsevier, New York, 1993). Nucleic acid molecules that hybridizeunder stringent conditions to a human ZAP-70 protein-encoding sequencewill typically hybridize to a probe based on either an entire humanZAP-70 protein-encoding sequence or selected portions of the encodingsequence under wash conditions of 2×SSC at 50° C.

Nucleic acid sequences that do not show a high degree of sequenceidentity may nevertheless encode similar amino acid sequences, due tothe degeneracy of the genetic code. It is understood that changes innucleic acid sequence can be made using this degeneracy to producemultiple nucleic acid molecules that all encode substantially the sameprotein.

Small interfering RNAs: Synthetic or naturally-produced small doublestranded RNAs (dsRNAs) that can induce gene-specific inhibition ofexpression in invertebrate and vertebrate species are provided. TheseRNAs are suitable for interference or inhibition of expression of atarget gene and comprise double stranded RNAs of about 15 to about 40nucleotides containing a 3′ and/or 5′ overhang on each strand having alength of 0- to about 5-nucleotides, wherein the sequence of the doublestranded RNAs is essentially identical to a portion of a coding regionof the target gene for which interference or inhibition of expression isdesired. The double stranded RNAs can be formed from complementaryssRNAs or from a single stranded RNA that forms a hairpin or fromexpression from a DNA vector.

Specific binding agent: An agent that binds substantially only to adefined target. Thus a protein-specific binding agent bindssubstantially only the specified protein. By way of example, as usedherein, the term “ZAP-70-protein specific binding agent” includesanti-ZAP-70 protein antibodies (and functional fragments thereof) andother agents (such as soluble receptors) that bind substantially only tothe ZAP-70 protein.

Anti-ZAP-70 protein antibodies may be produced using standard proceduresdescribed in a number of texts, including Harlow and Lane (Antibodies, ALaboratory Manual, CSHL, New York, 1988). The determination that aparticular agent binds substantially only to the specified protein mayreadily be made by using or adapting routine procedures. One suitable invitro assay makes use of the Western blotting procedure (described inmany standard texts, including Harlow and Lane (Antibodies, A LaboratoryManual, CSHL, New York, 1988)). Western blotting may be used todetermine that a given protein binding agent, such as an anti-ZAP-70protein monoclonal antibody, binds substantially only to the ZAP-70protein.

Shorter fragments of antibodies can also serve as specific bindingagents. For instance, Fabs, Fvs, and single-chain Fvs (SCFvs) that bindto a specified protein would be specific binding agents. These antibodyfragments are defined as follows: (1) Fab, the fragment which contains amonovalent antigen-binding fragment of an antibody molecule produced bydigestion of whole antibody with the enzyme papain to yield an intactlight chain and a portion of one heavy chain; (2) Fab′, the fragment ofan antibody molecule obtained by treating whole antibody with pepsin,followed by reduction, to yield an intact light chain and a portion ofthe heavy chain; two Fab′ fragments are obtained per antibody molecule;(3) (Fab′)2, the fragment of the antibody obtained by treating wholeantibody with the enzyme pepsin without subsequent reduction; (4)F(ab′)₂, a dimer of two Fab′ fragments held together by two disulfidebonds; (5) Fv, a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; and (6) single chain antibody (“SCA”), agenetically engineered molecule containing the variable region of thelight chain, the variable region of the heavy chain, linked by asuitable polypeptide linker as a genetically fused single chainmolecule. Methods of making these fragments are routine.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and non-human mammals.

Transformed: A transformed cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. As used herein,the term transformation encompasses all techniques by which a nucleicacid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergenes and other genetic elements known in the art.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Hence “comprisingA or B” means include A, or B, or A and B. It is further to beunderstood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

III Overview of Several Embodiments

The inventors have discovered that ZAP-70 is expressed at high levels inthe B-cells of CLL/SLL patients, and more particularly in the subset ofCLL which tends to have a more aggressive clinical course that isespecially in CLL/SLL patients with unmutated Ig genes. Because of thecorrelation between ZAP-70 expression levels and Ig gene mutationstatus, ZAP-70 can be used as a prognostic indicator to identify thosepatients likely to have severe disease (high ZAP-70, unmutated Iggenes), and who are therefore candidates for aggressive therapy.

Detecting ZAP-70 protein expression, for instance by Western blotting,immunohistochemistry, flow cytometry, or immunofluorescence, can serveas easily performed assays to distinguish the two groups of CLLpatients. Further, ZAP-70 is useful as a target for therapeuticstrategies, either directly or as an indicator of a type of CLL thatresponds to certain treatments.

In another embodiment, ZAP-70 RNA levels can be used similarly to ZAP-70protein levels.

One embodiment is a method of detecting a biological conditionassociated with ZAP-70 overexpression in a subject, which methodinvolves determining whether the subject has ZAP-70 nucleic acid orZAP-70 protein overexpression, wherein the biological conditioncomprises Ig-unmutated CLL. Examples of this method are methods ofpredicting a predisposition to poor clinical outcome in a subject. Suchmethods involve determining whether the subject overexpresses ZAP-70protein, wherein presence of ZAP-70 protein overexpression indicates thepredisposition to poor clinical outcome.

Specific examples of the methods of detecting a biological conditionassociated with ZAP-70 involve reacting at least one ZAP-70 moleculecontained in a sample (e.g., one containing a neoplastic cell) from thesubject with a reagent comprising a ZAP-70-specific binding agent toform a ZAP-70:agent complex. In examples of these methods, the ZAP-70molecule is a ZAP-70 encoding nucleic acid or a ZAP-70 protein. TheZAP-70 specific binding agent is, in some embodiments, a ZAP-70oligonucleotide or a ZAP-70 protein specific binding agent.

In another embodiment, the method further involves in vitro amplifying aZAP-70 nucleic acid prior to detecting the abnormal ZAP-70 nucleic acid.By way of example, the ZAP-70 nucleic acid is in vitro amplified usingat least one oligonucleotide primer derived from a ZAP-70-proteinencoding sequence. Examples of such oligonucleotide primers comprise atleast 15 contiguous nucleotides from SEQ ID NO: 1.

Another embodiment is a method of detecting a biological conditionassociated with ZAP-70 overexpression in a subject, wherein the ZAP-70molecule is a ZAP-70 encoding sequence. In such methods, the bindingagent is optionally a labeled nucleotide probe. For instance, examplesof such nucleotide probes have a sequence selected from the groupconsisting of: SEQ ID NO: 1; nucleic acid sequences having at least 85%sequence identity with SEQ ID NO: 1; and fragments thereof at least 15nucleotides in length.

Another embodiment is a method of detecting a biological conditionassociated with ZAP-70 overexpression in a subject, wherein the ZAP-70molecule is a ZAP-70 protein. In representative examples of suchmethods, the complexes are detected by Western blot assay, or by ELISA.By way of example, the ZAP-70 protein in such methods may include asequence selected from the group consisting of: SEQ ID NO: 2; amino acidsequences having at least 85% sequence identity with SEQ ID NO: 2; andconservative variants thereof.

In a further embodiment is a method of treating a subject overexpressingZAP-70, wherein the method involves administering to the subject atherapeutically effective amount of an agent that inhibits ZAP-70function or expression. In specific examples, the agent inhibits ZAP-70expression or ZAP-70 function. In other specific examples, the agent isan oligonucleotide that is homologous to a nucleic acid sequence as setforth as SEQ ID NO: 1. The agent can also be a kinase inhibitor or adrug that affects the ability of ZAP-70 to interact with other proteins.Such methods involve treating subjects for Ig-unmutated chroniclymphocytic leukemia associated with ZAP-70 overexpression.

In still further examples, the ZAP-70-specific binding agent is aZAP-70-specific antibody (e.g., a monoclonal antibody) or a functionalfragment thereof.

Also provided herein are kits for detecting overexpression of ZAP-70protein in a subject (such as a mammal, for instance a human). Examplesof such kits comprising a ZAP-70 protein specific binding agent, forinstance a specific binding agent is capable of specifically binding toan epitope within the amino acid sequence shown in SEQ ID NO: 2; aminoacid sequences that differ from those specified in SEQ ID NO: 2 by oneor more conservative amino acid substitutions; amino acid sequenceshaving at least 85% sequence identity to; or antigenic fragments of inof these.

Still further example kits include a means for detecting binding of theZAP-70 protein binding agent to a ZAP-70 polypeptide.

Specific examples of provided kits include as part of the kit an amountof a ZAP-70 protein binding agent, and the agent is an antibody.

Another embodiment is a kit for determining whether or not a subject hasa biological condition associated with ZAP-70 overexpression, bydetecting an overabundance of ZAP-70 protein or nucleic acid in a sampleof tissue and/or body fluids from the subject. Examples of this kitinclude as elements of the kit a container comprising an antibodyspecific for ZAP-70 protein or an oligonucleotide homologous to a ZAP-70nucleic acid; and instructions for using the kit, the instructionsindicating steps for: performing a method to detect the presence ofZAP-70 protein or nucleic acid in the sample; and analyzing datagenerated by the method, wherein the instructions indicate thatoverabundance of ZAP-70 protein or nucleic acid in the sample indicatesthat the individual has or is predisposed to the biological condition.Optionally, such kits may further include a container that comprises adetectable antibody capable of binding to the ZAP-70 protein specificantibody or a container that comprises a labeled nucleotide probecapable of specifically hybridizing to the ZAP-70 nucleic acid.

A still further embodiment is an in vitro assay kit for determiningwhether or not a subject has a biological condition associated with anabnormal ZAP-70 expression. Such kits include a container comprising aZAP-70 protein specific antibody or an oligonucleotide homologous to aZAP-70 nucleic acid; a container comprising a negative control sample;and instructions for using the kit, the instructions indicating stepsfor: performing a test assay to detect a quantity of ZAP-70 protein ornucleic acid in a test sample of tissue and/or bodily fluid from thesubject, performing a negative control assay to detect a quantity ofZAP-70 protein or nucleic acid in the negative control sample; andcomparing data generated by the test assay and negative control assay,wherein the instructions indicate that a quantity of ZAP-70 protein ornucleic acid in the test sample more than the quantity of ZAP-70 proteinor nucleic acid in the negative control sample indicates that thesubject has the biological condition. Optionally, such kits may furtherinclude a container that comprises a detectable antibody capable ofbinding to the ZAP-70 protein specific antibody or a container thatcomprises a labeled nucleotide probe capable of specifically hybridizingto the ZAP-70 nucleic acid.

In specific examples of the provided kits, the biological conditionassociated with abnormal ZAP-70 expression (e.g., overexpression) is Igunmutated-CLL.

Also provided are methods of modifying a level of expression of a ZAP-70protein in a subject in order to reduce, ameliorate, or control CLL,which method involves expressing in the subject a recombinant geneticconstruct comprising a promoter operably linked to a nucleic acidmolecule, wherein the nucleic acid molecule comprises at least 15consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO:1, or a sequence at least 85% identical to SEQ ID NO: 1, and expressionof the nucleic acid molecule changes expression of the ZAP-70 protein.In examples of such methods, the nucleic acid molecule is in antisenseorientation relative to the promoter.

IV. ZAP-70

ZAP-70 (GenBank Accession no. XM_(—)047776) is a member of theZAP-70/Syk family of protein tyrosine kinases. ZAP-70 is expressed inT-cells and natural killer cells, while Syk is present in mosthematopoietic cells, including B cells, mast cells, immature T cells,and platelets. ZAP-70 and Syk are structurally similar, and theircellular functions may partially overlap (Zhang and Siraganian, J.Immun. 163:2508-2516, 1999).

ZAP-70 associates with the zeta (t) subunit of the T-cell antigenreceptor (TCR) (Chan et al., Cell 71:649-662, 1992). It undergoestyrosine phosphorylation and is essential in mediating signaltransduction following TCR stimulation. Similarly, Syk is essential inmediating B-cell responses to antigen.

It has been reported that ZAP-70 phosphorylation activity is reduced orundetectable in malignant cells of cutaneous T-cell lymphoma (Fargnoliet al., Leukemia 11(8)1338-1346, 1997). ZAP-70 is not known to beexpressed in B-cells.

Provided herein is the identification of ZAP-70 RNA expression levels asa prognostic marker of CLL; this has been reported in Rosenwald et al.,J. Exp. Med. 194:1639-1647, 2001. Also provided herein is theidentification of ZAP-70 protein levels as a prognostic marker of CLL.Based on mRNA and protein expression levels in samples from patientssuffering CLL, ZAP-70 has been identified as a molecule that can be usedin the clinical classification of patients with CLL/SLL.

V. ZAP-70 Expression to Guide Therapy in CLL

Currently, there is no curative treatment for CLL/SLL and therapy isdelayed for as long as possible (NCI trial lists, JNCI 1999). Only whenclinical symptoms become severe enough is treatment initiated. With theadvent of newer therapies it might be beneficial to start treatmentbefore symptoms appear if there was a reliable method to identifypatients who would have early disease progression and a more aggressiveclinical course (Byrd, Sem in Oncol, 1998). ZAP-70 may be able to guidesuch a strategy of risk adapted treatment. Specifically ZAP-70 negativepatients might best be managed by a watch and wait strategy and would bespared potentially harmful treatment. On the other hand, ZAP-70 positivepatients might benefit from early intervention before a large tumor bulkaccumulates and the patients are weakened by progressive disease.Furthermore, because ZAP-70 expression characterizes CLL cells with adistinct biology it may be possible to select patients for targetedtherapeutic strategies based on ZAP-70 expression.

VI. A Role for Zap-70 in the Pathogenesis of CLL/SLL

ZAP-70 is a tyrosine kinase, which associates with the T cell receptorand plays a pivotal role in T cell activation and development.Overexpression or constitutive activation of tyrosine kinases has beendemonstrated to be critically involved in a number of malignanciesincluding leukemias and several types of solid tumors. Thus, thedetection of ZAP-70 protein expression in CLL/SLL cells raises thequestion of a pathogenetic role of this kinase in the development orpropagation of CLL/SLL. Given the relatively slow growth rate of CLLcells it is not surprising that evidence has not been detected for aconstitutive activation of the ZAP-70 kinase in CLL blood cells.However, it is conceivable that activation of ZAP-70 occurs in the bonemarrow or lymphatic organs. CLL cells receive survival signals fromstromal cells in the microenvironment of these sites. Similarly,microarray data indicate that CLL cells receive important activationsignals through the antigen receptor.

The presence of ZAP-70 might affect sensitivity to, duration and/orintensity of such signals and could thus be a key factor for the moreaggressive form of CLL. Therefore, targeting ZAP-70 in CLL patientscould benefit especially the patients with the more rapidly progressiveform of the disease. Because of the important role of ZAP-70 in T-cellsignaling, interest in inhibitors targeting ZAP-70 has been high andseveral candidate drugs have been designed and found to inhibit ZAP-70function in preclinical models (Nishikawa, Mol Cell 2000). The clinicalfocus of such drugs has been perceived to be immunosuppression. However,as outlined above ZAP-70 might be a promising target for anti-leukemictherapy in CLL. Furthermore, drugs targeting ZAP-70 might have activityin lymphomas, given that ZAP-70 expression was detected in severallymphoma cell lines. Further studies to elucidate these interactionswill include functional studies, the use of inhibitors to analyzechanges in gene expression profile and the analysis of gene expressionin CLL cells under different physiologic conditions.

ZAP-70 will find immediate use as a prognostic marker in CLL/SLL andwill be helpful to guide treatment strategies. Even more intriguing isits potential role in disease pathogenesis and progression and thepossibility that inhibitors of ZAP-70 may lead to targeted therapy ofCLL.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the invention to the particular features or embodiments described.

EXAMPLES Example 1 Gene Expression Profiling of B Cell ChronicLymphocytic Leukemia

The most common human leukemia is B cell chronic lymphocytic leukemia(CLL), a malignancy of mature B cells with a characteristic clinicalpresentation but a variable clinical course. The rearrangedimmunoglobulin (Ig) genes of CLL cells may be either germ-line insequence or somatically mutated. Lack of Ig mutations (UM-CLL) defined adistinctly worse prognostic group of CLL patients raising thepossibility that CLL comprises two distinct diseases.

Using genomic-scale gene expression profiling, it is demonstrated thatCLL is characterized by a common gene expression “signature,”irrespective of Ig mutational status, suggesting that CLL cases share acommon mechanism of transformation and/or cell of origin. Nonetheless,the expression of hundreds of other genes correlated with the Igmutational status, including many genes that are modulated in expressionduring mitogenic B cell receptor signaling. These genes were used tobuild a CLL subtype predictor for use in the clinical classification ofpatients with this disease.

The observation that the rearranged Ig variable genes in CLL cells areeither unmutated (UM-CLL) or mutated (M-CLL) indicated that CLL cancomprise two different diseases lumped together using standarddiagnostic methods. Somatic hypermutation of Ig genes is a specializeddiversification mechanism that is activated in B cells at the germinalcenter stage of differentiation. Thus, it was indicated that CLLincludes two disparate malignancies, one derived from an Ig-unmutated,pregerminal center B cell, and the other from an Ig-mutated B cell thathas passed through the germinal center. This “two disease” model of CLLwas further supported by the observation that Ig-unmutated andIg-mutated CLL patients had distinctly different clinical courses.

One model indicates that Ig-unmutated and Ig-mutated CLL are not highlyrelated to each other in gene expression. A precedent for this model isfound in the recent demonstration that another lymphoid malignancy,diffuse large B cell lymphoma (DLBCL), actually includes two distinctdiseases that are morphologically indistinguishable but which havelargely nonoverlapping gene expression profiles. Alternatively, allcases of CLL can have a common cellular origin and/or a common mechanismof malignant transformation. Thus, in this model, Ig-mutated andIg-unmutated CLL cases share a gene expression signature that ischaracteristic of CLL.

To demonstrate these two models, and to identify molecular differencesbetween CLL patients that might influence their clinical course, thegene expression phenotype of CLL on a genomic scale was determined usingLymphochip cDNA microarrays (Alizadeh et al., Nature 403:503-511, 2000;Alizadeh et al., Cold Spring Harbor Symp. Quant. Biol. 64:71-78, 1999).The data demonstrate that CLL, irrespective of the Ig mutational status,is defined by a characteristic gene expression signature, thus favoringthe notion that all cases share some aspects of pathogenesis.Nonetheless, hundreds of genes were found to be differentially expressedbetween Ig-unmutated and Ig-mutated CLL, providing the first molecularinsight into the biological mechanisms that lead to the divergentclinical behaviors of these subgroups of CLL patients. The unexpectedfinding that B cell activation genes were differentially expressedbetween the two Ig-mutational subgroups in CLL indicates that signalingpathways downstream of the B cell receptor (BCR) contribute to the moreaggressive clinical behavior of the Ig-unmutated subtype.

Methods and Materials Microarray Procedures.

Peripheral blood samples from CLL patients diagnosed according toNational Cancer Institute guidelines (Cheson et al., Blood 87:4990-4997,1996) were obtained after informed consent and were treated anonymouslyduring microarray analysis. Thirty-three CLL patients studied had notreceived chemotherapy at the time of sample acquisition and fourpatients had received prior treatment. Ig mutational status was onlystudied in untreated patients. Leukemic cells from CLL blood sampleswere purified by magnetic selection for CD19+ (Miltenyi Biotec) at 4° C.before mRNA extraction and microarray analysis. Other mRNA samples fromnormal and malignant lymphoid populations have been describedpreviously, as have cell purification methods and array methods(Alizadeh et al., Nature 403:503-511, 2000). All microarray experimentsused the Cy5 dye to generate the experimental cDNA probe from mRNA ofnormal and malignant lymphocytes, and the Cy3 dye to generate thereference cDNA probe from mRNA pooled from nine lymphoma cell lines asdescribed previously (Alizadeh et al., Nature 403:503-511, 2000).

Initial microarray data selection was based on fluorescence signalintensity. Each selected data point either had 100 relative fluorescentunits (RFU's) above background in both the Cy3 and Cy5 channels, or 500RFU's above background in either channel alone. A supervised selectionof genes preferentially expressed in CLL cells was performed as follows.First, it was determined that the majority of cell lines that were usedto construct the reference pool of mRNA were derived from DLBCL. Thepercentage of CLL samples with expression ratio >3 relative to thereference cell line pool was calculated, and the same calculation wasalso performed for the DLBCL samples. Genes were selected for which >50%of the CLL samples, and <25% of the DLBCL samples, had ratios >3.Additionally, genes were selected if the average CLL ratio was greaterthan the average DLBCL ratio by greater than threefold. Representativegenes were chosen by computing the average expression in CLL samples andthe average expression in resting B cell samples (adult and cord blood Bcells). CLL signature genes were chosen to be at least twofold morehighly expressed in CLL than in resting B cells and CLL/resting B cellgenes were chosen to be expressed equivalently (within twofold) in thetwo sample sets. Duplicate array elements representing the same geneswere removed. Germinal center genes were chosen from a previous analysis(Alizadeh et al., Nature 403:503-511, 2000).

RT-PCR.

500 ng poly-A+ mRNA was used to generate first strand cDNA usingSuperscript (Life Technologies) together with random hexamers andoligo-dT primers. ZAP-70 oligonucleotide primers (5′ TCTCCAAAGCACTGGGTG3′, SEQ ID NO: 3; 5′ AGCTGTGTGTGGAGACAACCAAG 3′, SEQ ID NO: 4) were thenused for PCR amplification for 27 cycles.

Statistical Analysis.

A two-group t-statistic on log2 expression ratios was used to measurethe ability of each array element to discriminate between the two CLLmutational subtypes univariately. For multivariate subtype prediction, alinear combination of log2 expression ratios for array elements thatwere significant at the P<0.001 significance level were used in theunivariate analysis. The expression ratios were weighted in the linearcombination by the univariate t statistics. The linear combination wascomputed for each sample and the average linear combination was computedfor each CLL subtype. The midpoint of the two CLL subtype means was usedas a cut-point for subtype prediction. For the cross-validationanalysis, the subtype predictor was calculated by sequentially omittingone sample from the test set of cases, and using the remaining cases togenerate the predictor. Calculation of the P value from the permutationdistribution of the t-statistic also demonstrated the high statisticalsignificance of the differential gene expression between the CLLsubtypes. Classification was determined on all CLL cases with theexception of CLL-60 (Ig-unmutated) and CLL-21 and CLL-51 (minimallymutated cases).

The choice of B cell activation genes was made as follows. The B cellactivation series of microarray experiments included several differentstimulations with anti-IgM for 6, 24, and 48 hours for each Lymphochiparray element, the data were averaged at each activation time point, andthen selected those elements that gave a twofold induction compared withthe resting B cell average for at least one time point.

Results The Gene Expression Signature of CLL.

Gene expression in CLL samples (n=37) was profiled using Lymphochip cDNAmicroarrays containing 17,856 human cDNAs (Alizadeh et al., Cold SpringHarbor Symp. Quant. Biol. 64:71-78, 1999). To facilitate comparison ofeach CLL mRNA sample with the others and with previously generated datasets, gene expression in each CLL mRNA sample was compared to a commonreference mRNA pool prepared from lymphoid cell lines (Alizadeh et al.,Nature 403:503-511, 2000; Alizadeh et al., Cold Spring Harbor Symp.Quant. Biol. 64:71-78, 1999). Using this strategy, the relative geneexpression in the CLL cases could be compared with other B cellmalignancies (DLBCL and follicular lymphoma) and of normal B cell and Tcell sub-populations. Expression data from 328 Lymphochip array elementsrepresenting 247 genes that were selected in a supervised fashion (seeMaterials and Methods) to be more highly expressed in the majority ofCLL samples than in DLBCL samples (n=40) were obtained. These genes fallinto two broad categories. Genes in the first category define a CLL geneexpression “signature” that distinguishes CLL from various normal B cellsubsets and from other B cell malignancies. The CLL signature genes werenot expressed highly in resting blood B cells or in germinal center Bcells. This group of genes includes several genes not previouslysuspected to be expressed in CLL (e.g., Wnt3, titin, Ror1) as well as anumber of novel genes from various normal and malignant B cell cDNAlibraries. By contrast, CLL cells lacked expression of most genes thatare preferentially expressed in germinal center B cells. In addition tothis set of CLL signature genes, CLL preferentially expressed a set ofgenes that distinguish resting, GO stage blood B cells frommitogenically activated blood B cells and germinal center B cells thatare traversing the cell cycle. The expression of these resting B cellgenes by CLL cells is consistent with the indolent, slowly proliferatingcharacter of this malignancy.

One of these resting B cell samples was prepared from human umbilicalcord blood that is enriched for B cells bearing the CD5 surface marker,a B cell subpopulation that has been proposed to be the normalcounterpart of CLL. The cord blood B cells were >80% CD5⁺ by FACS®analysis whereas resting B cells from adult blood are 10-20% CD5⁺(Geiger et al., Eur. J. Immunol., 30:2918, 2000). Notably higherexpression of the CLL signature genes in the cord blood B cell samplethan in the adult B cell sample was not observed, and no overallcorrelation in the expression of genes was observed between CLL andeither adult or cord blood B cells (Pearson correlation coefficients−0.27 and −0.21, respectively). Thus, the gene expression profilinganalysis does not provide support for the hypothesis that the CD5⁺ Bcell is a CLL precursor. However, that the expression of the CLLsignature genes can be due to the oncogenic mechanisms of CLL andtherefore is not a feature of any normal B cell subpopulation.

Ig Mutational Status.

The expressed Ig heavy chain genes were sequenced from 28 CLL cases andcompared with known germ-line encoded Ig VH segments as describedpreviously (Bessudo et al., Blood 88:252-260, 1996). By convention, VHsequences that matched known germ line sequences with >98% identity wereconsidered unmutated, as any minor differences observed in this groupwere assumed to reflect genetic polymorphism (Fais et al., J. Clin.Invest. 102:1515-1525, 1998; Hamblin et al., Blood 94:1848-1854, 1999;Damle et al., Blood 94:1840-1847, 1999). By this criterion, 16 CLL casesin the study set were unmutated. The remaining cases were furtherseparated into a group of 10 highly mutated cases (<97% identity withany germ-line VH segment) and a group of two cases that were minimallymutated (>97% but <98% identity with known germ-line VH genes). CLLcases were grouped according to Ig mutational status as indicated above.Although some variation in expression of the CLL signature andCLL/resting B cell genes was evident between CLL patients, most patientsin each Ig mutational subtype highly expressed these genes at comparablelevels. Furthermore, an unsupervised hierarchical clustering of the CLLcases using 10,249 Lymphochip array elements resulted in a clusteringdendrogram in which the Ig-unmutated and Ig-mutated CLL cases wereextensively intermingled. Thus, the overall gene expression profiles ofthe two CLL subtypes were largely overlapping.

Segregation of the patients according to Ig mutational status revealedthat Ig-unmutated CLL patients had a significantly worse clinicalcourse, requiring earlier treatment, than the Ig-mutated CLL patients,in keeping with previous reports (Hamblin et al., Blood 94:1848-1854,1999; Damle et al., Blood 94:1840-1847, 1999).

CLL Subtype Distinction Genes.

Given the dramatically different clinical behavior of the Ig-unmutatedand Ig-mutated CLL patients, gene expression differences can bediscerned between these groups. To both demonstrate such genes andstatistically validate their relationship to the Ig-mutationalsubgroups, the Ig mutational analysis was conducted independently andsequentially in two random subsets of the CLL patients (FIG. 1). The“training” set consisted of ten Ig-unmutated cases and eight Ig-mutatedcases. In this gene discovery phase, the minimally mutated CLL caseswere assigned to the mutated class. The mean expression of each gene wasthen calculated for both mutational subgroups and the statisticalsignificance of the difference of these means was determined. All genesthat discriminated between the mutational subgroups at a significance ofP<0.001 (n=56) were used to form a “predictor” that could be used toassign a CLL sample to a mutational subgroup based on gene expression(see Methods).

The performance of this CLL subtype predictor was initially demonstratedusing a cross-validation strategy (FIG. 1A). One of the 18 CLL samplesin the training set was omitted, the statistically significant geneswere determined, and a predictor was calculated based on the remaining17 samples. The omitted sample was then assigned to a CLL subtype basedon gene expression using this predictor. The Ig mutational status of 17CLL samples was correctly assigned by this procedure with onemisassignment. To determine the statistical significance of this result,1,000 random permutations of the assignments of CLL samples to the Igmutation subgroups were created. For each permutation, thecross-validation process described above was repeated. Only one of the1,000 random permutations generated a predictor that performed as wellas the predictor based on the unpermutated data, demonstrating that thesignificance of the gene expression difference between the CLL subtypeswas P=0.001.

Finally, the Ig mutational status of a “test” set of 10 additional CLLcases was determined and the predictor derived from the training set wasused to assign the cases in this test set to a CLL subtype based on geneexpression in a blinded fashion (FIG. 1B). Nine out of ten of the testcases were correctly assigned, showing the ability of the CLL subtypepredictor to correctly assign new CLL cases based on gene expressiondata that was not used to generate the predictor. The one misclassifiedCLL case (CLL-60) clearly was an outlier in gene expression. Takentogether with the cross-validation results, these data demonstrate thatgene expression can define CLL subtypes that have different degrees ofIg mutation.

These findings can be used to create a diagnostic test for the CLLsubtypes based upon gene expression. In this regard, one of the mostdifferentially expressed genes from the analysis of the training set ofcases, ZAP-70, could classify all of the cases in both the training andthe test set with 100% accuracy. Likewise, predictors based on two genes(ZAP-70 and IM1286077) or three genes (ZAP-70, IM1286077,activation-induced C-type lectin) discovered using the training setformed CLL subtype predictors that performed with 100% accuracy on thetraining set and test set of CLL cases.

The search for CLL subtype distinction genes was next expanded usingdata from both the training set and test set of CLL cases. The two CLLcases with minimal Ig mutations (CLL-22 and CLL-51) were excluded basedon the possibility that their Ig sequences might actually represent asyet undescribed polymorphic VH alleles. CLL-60 was excluded based on itsunusual gene expression characteristics that led to itsmisclassification by the CLL subtype predictor. Two hundred and fiveLymphochip array elements (175 genes) that were differentially expressedbetween the CLL subtypes had a statistical significance of P<0.001.Hierarchical clustering of the CLL cases based on expression of thesegenes placed the majority of Ig-unmutated CLL cases in one cluster andthe Ig-highly mutated CLL cases in another. As expected, CLL-60 was moreclosely aligned with the Ig-mutated CLL cases, though it was an outlierfrom the major cluster of Ig-mutated CLL cases. Interestingly, both ofthe CLL cases with a low Ig mutational load were also outliers, thoughthey were more closely related to the Ig-mutated CLL subtype than to theIg-unmutated CLL subtype. These data define two predominant CLL subtypesthat differ in the expression of hundreds of genes but also demonstratethat additional minor CLL subtypes may exist that have distinct geneexpression profiles. ZAP-70 was the most tightly discriminating gene,with an average 4.3-fold higher expression in Ig-unmutated CLL than inIg-mutated CLL (P<10-6). RT-PCR analysis confirmed ZAP-70 expression intwo Ig-unmutated CLL cases (CLL-48 and CLL-49), in contrast to CLL-66and CLL-69 that were Ig-mutated. Surprisingly, ZAP-70 expression wasalso observed in several B cell lines (LILA, LK-6, OCI-Ly2).

Relationship Between B Cell Activation and the CLL Subtype Distinction.

Several of the CLL subtype distinction genes are known or suspected tobe induced by protein kinase C (PKC) signaling, includingactivation-induced C-type lectin (Hamann et al., Immunogenetics, 45:295,1997), MDS019, a very close paralogue of phorbolin 1 (Madsen et al., J.Invest. Dermatol. 113:162-169, 1999), and gravin, a scaffold proteinthat binds PKC and may regulate its activity (Nauert et al., Curr. Biol.7:52-62, 1997). One mechanism by which PKC is activated in B cells isthrough BCR signaling (Cambier et al., Annu. Rev. Immunol. 12:457-486,1994). Therefore, it was determined whether the CLL subtype distinctiongenes are regulated during activation of blood B cells, using a geneexpression database generated previously using Lymphochip microarrays(Alizadeh et al., Nature 403:503-511, 2000). Strikingly, many of thegenes that were more highly expressed in Ig-unmutated CLL were inducedduring activation of blood B cells. Many of these genes encode proteinsinvolved in cell cycle control (e.g., cyclin D2) or in cellularmetabolism required for cell cycle progression (e.g., HPRT and othernucleotide modifying enzymes). Conversely, the majority of the genesthat were expressed at lower levels in Ig-unmutated CLL were stronglydown-modulated during B cell activation. These results demonstrate thatthe CLL subtype distinction genes are enriched for genes that aremodulated in expression by B cell activation. Indeed, 47% of the CLLsubtype distinction genes were induced during B cell activation, whereasonly 18% of all Lymphochip genes were in this category.

Gene Expression in CLL Provides New Understanding of the Etiology of CLLand the Divergent Clinical Courses of Patients Suffering from CLL.

Using genomic-scale gene expression profiling, a current controversy inCLL pathogenesis, namely whether this diagnosis comprises more than onedisease entity, was addressed. CLL patients have been subdivided basedon the Ig mutational status of their leukemic cells (Fais et al., J.Clin. Invest. 102:1515-1525, 1998; Hamblin et al., Blood 94:1848-1854,1999; Damle et al., Blood 94:1840-1847, 1999), but it was unclearwhether these patients had molecularly distinct diseases. The datademonstrate that all CLL patients share a characteristic gene expressionsignature in their leukemic cells.

These findings support a model in which all cases of CLL have a commoncell of origin and/or a common mechanism of malignant transformation. Inthis model, the CLL-specific gene expression signature might representthe gene expression signature of a common normal precursor cell or itmight reflect the downstream gene expression consequences of a commononcogenic event. These findings are in contrast to the previousobservation that DLBCL consists of two disease entities that did nothave overlapping gene expression outside of genes involved inproliferation and in the host response to the tumor (Alizadeh et al.,Nature 403:503-511, 2000).

CLL cells proliferate slowly in vivo, driven by unknown signals.Therefore, it is notable that Wnt-3 was highly, and selectively,expressed in CLL. The Wnt gene family encodes secreted proteins thatsignal through cell surface receptors of the frizzled family to controldevelopment and mediate malignant transformation (Polakis, Genes Dev.14:1837-1851, 2000). Intriguingly, another CLL signature gene, Ror1,encodes a receptor tyrosine kinase with an extracellular domain thatresembles a Wnt interaction domain of frizzled (Saldanha et al., ProteinSci. 7:1632-1635, 1998). Recently, Wnt-3 has been shown to promoteproliferation of mouse bone marrow pro-B cells by initiating signalingevents leading to transcriptional activation by LEF-1 (Reya et al.,Immunity 13:15-24, 2000). Thus, CLL cells can use an autocrine mechanismof proliferation that is used normally by B cell progenitors.

It was nevertheless also found that the expression of hundreds of othergenes correlated with the Ig mutational status in CLL, providinginsights into the biological mechanisms that lead to the divergentclinical behaviors of CLL patients. The most differentially expressedgene between the CLL subtypes was ZAP-70, a kinase that transducessignals from the T cell antigen receptor, and is preferentiallyexpressed in normal T lymphocytes (Chu et al., Immunol. Rev.165:167-180, 1998). Differential expression of ZAP-70 between CLLsubtypes was therefore surprising, since its expression in normal Bcells has not been previously reported. However, by microarray analysisand RT-PCR analysis it was found that ZAP-70 mRNA is highly expressed insome B lymphoma cell lines along with being differentially expressed bythe CLL subtypes. A ZAP-70-related kinase, syk, transduces signals fromthe B cell receptor (BCR) (Turner et al., Immunol. Today 21:148-154,2000), raising the possibility that ZAP-70 might alter BCR signaling inCLL cells.

Another CLL subtype distinction gene, Pak1, could contribute to theresistance of CLL cells to apoptosis by phosphorylating Bad and therebypreventing Bad from inhibiting BCL-2 (Schurmann et al., Mol. Cell. Biol.20:453-461, 2000). Fibroblast growth factor receptor (FGFR) 1 is areceptor tyrosine kinase that can stimulate cellular proliferation afterinteraction with fibroblast growth factors. The higher expression ofFGFR1 in Ig-unmutated CLL is intriguing given that CLL patients haveelevated blood levels of basic fibroblast growth factor, which canactivate FGFR1 and block apoptosis in CLL (Aguayo et al., Blood96:2240-2245, 2000; Menzel et al., Blood 87:1056-1063, 1996).

Intriguingly, CLL subtype distinction genes were enriched for genes thatare modulated in expression during signaling of B cells through the BCR.One hypothesis raised by this observation is that the leukemic cells inIg-unmutated CLL may have ongoing BCR signaling. Interestingly, the VHrepertoire usage in the Ig-unmutated and Ig-mutated CLL is distinct(Fais et al., J. Clin. Invest. 102:1515-1525, 1998; Hamblin et al.,Blood 94:1848-1854, 1999; Damle et al., Blood 94:1840-1847, 1999) andthe combinations of VH, DH, and JH gene segments rearranged in CLL cellsare not random (Fais et al., J. Clin. Invest. 102:1515-1525, 1998;Hamblin et al., Blood 94:1848-1854, 1999; Damle et al., Blood94:1840-1847, 1999; Widhopf and Kipps, J. Immunol. 166:95-102, 2001;Johnson et al., J. Immunol. 158:235-246, 1997). These observationssuggest that the surface Ig receptors of CLL cells may have specificityfor unknown environmental or self-antigens. Indeed, CLL cells have beenshown to frequently produce antibodies that bind classical autoantigens(Borche et al., Blood 76:562-569, 1990; Sthoeger et al., J. Exp. Med.169:255-268, 1989; Broker et al., J. Autoimmun. 1:469-481, 1988). Thegene expression profiling data presented herein indicate thatIg-unmutated CLL cells is continuously stimulated in vivo by antigen,giving rise to a gene expression profile that is consistent with BCRsignaling. Indeed, CLL cells from patients with progressive disease weremore readily stimulated by BCR cross-linking to synthesize DNA than wereCLL cells from patients with stable disease (Aguilar-Santelises et al.,Leukemia 8:1146-1152, 1994). Although this study did not distinguishbetween Ig-unmutated and Ig-mutated CLL, the results are consistent witha differential ability of these subtypes to signal through the BCR.Alternatively, Ig-unmutated CLL cells can activate the same signalingpathways that are engaged during B cell activation as a result ofgenetic changes in the leukemic cells or by other pathologicalmechanisms.

An immediate clinical application of these results is the differentialmolecular diagnosis of CLL. It was demonstrated that as few as 1-3 genescould correctly assign patients to a CLL subtype with 100% accuracy.Thus, the results can be used to establish a variety of prognostictests. Examples of such tests include RNA- and DNA-based techniques suchas microarrays or PCR. A prognostic test, such as a quantitative RT-PCRtest to diagnose the CLL subtypes, would be easier to adopt clinicallythan DNA sequence analysis of Ig variable regions. Given the relativelybenign course of Ig-mutated CLL, a simple diagnostic test based on geneexpression provides valuable prognostic information for CLL patients andcan be used to guide treatment decisions.

In addition, new therapeutic approaches to this currently incurableleukemia are provided herein. First, the protein products of some of theCLL signature genes present new targets for therapeutic agents or drugs.These agents can include kinase inhibitors, antibodies for use in mAbtherapy, and molecular decoys that affect protein-protein interactions(for example, antagonists). The protein products may also be of use invaccine approaches to CLL. Agents that can modify the expression of theprotein products of the CLL signature genes include antisenseoligonucleotides or small inhibitory RNA. Second, the unexpected findingthat B-cell activation genes were upregulated in Ig-unmutated CLLpatients indicates that signaling pathways downstream of the BCRcontribute to the more progressive clinical course of these patients.Thus, therapeutic targeting of these signaling pathways, using methodssuch as those described above, will specifically benefit those CLLpatients that show gene expression evidence that these pathways areactive.

Example 2 ZAP-70 Expression Identifies a CLL/SLL Subtype

Given the clinical differences between Ig-mutated and Ig-unmutated CLL,it would be beneficial to incorporate this distinction into the clinicaldiagnosis of CLL patients. Most clinical diagnostic laboratories do nothave the routine ability to sequence IgVH genes. This analysis is timeconsuming and expensive, making it doubtful that it can be establishedas a clinical test available to all CLL patients. Further, thedistinction between Ig-mutated and Ig-unmutated CLL is based on thedegree of identity between the CLL IgVH sequence and the closestgermline IgVH sequence. However, the optimal cut point for thisdistinction is not clear. Early studies used a cutoff of 98% sequenceidentity to allow for germline Ig polymorphisms in the human population(Damle et al., Blood, 94:1840, 1999; Hamblin et al., Blood, 94:1848,1999; Oscier et al., Blood, 89:4153, 1997). In a recent study with 300patients, a cutoff of 97% sequence identity was optimal fordistinguishing CLL patients that had different overall survival rates(Krober et al., Blood, 100:1410, 2002). However, the 95% confidenceinterval for this distinction ranged from 96% to 98% sequence identity.

Expression of CD38, as determined by flow cytometry, has been shown tohave prognostic significance in CLL (D'Arena et al., Leuk. Lymphoma,42:109, 2001; Del Poeta et al., Blood, 98:2633, 2001' Durig et al.,Leukemia, 16:30, 2002; Ibrahim et al., Blood, 98:181, 2001). Initially,it was proposed that CD38 might serve as a surrogate marker for IgVHmutational status (Damle et al., Blood, 94:1840, 1999). Subsequentstudies have not always shown this relationship (Hamblin et al., Blood,99:1023, 2002; Thunberg et al., Blood, 97:1892, 2001). It has also beensuggested that CD38 expression might add to the prognostic informationin patients with known IgVH status (Hamblin et al., Blood, 99:1023,2002), but two large studies that together included more than 500patients failed to confirm CD38 as an independent prognostic factor inmultivariate analysis (Krober et al., Blood, 100:1410, 2002; Oscier etal., Blood, 100:1177, 2002). Some of the differences may be due totechnical aspects of the CD38 assays and the choice of an optimal cutpoint for the number of CD38+ cells. The largest study to date foundthat a cutoff of 7% was best at separating different prognostic groups(Krober et al., Blood, 100:1410, 2002). Another confounding issue isthat CD38 expression by the leukemic clone may change during the courseof the disease, and an increase of CD38 expression may herald diseaseprogression (Hamblin et al., Ann. Hematol., 81:299, 2002).

Here, in a separate study from that described in Example 1, it isdemonstrated how ZAP-70 expression is able to discriminate betweenclinical subgroups of CLL/SLL. ZAP-70 expression correlates withunmutated immunoglobulin genes and more aggressive disease. Thus, theresults of this study confirm and expand the results of the studydescribed in Example 1. Tests were developed to assess ZAP-70 nucleicacid and ZAP-70 protein expression suitable for routine clinicallaboratory use. Thus, testing for ZAP-70 expression can be performed inthe clinic, to yield important prognostic information and help guidetreatment decisions.

Materials and Methods Patient Samples.

All patients included in this study were enrolled in a clinical protocolat the National Institutes of Health and gave informed consent to theuse of blood and tissue samples for research. Peripheral bloodmononuclear cells (PBMC) were obtained by ficoll gradient centrifugation(ICN Biomedicals). Leukemic cells were purified by magnetic selectionfor CD19 expression (Miltenyi Biotech). To obtain paraffin cell pellets,PBMC were washed in PBS, pelleted and resuspended in plasma. Clotformation was initiated with the addition of thrombin. The clot wasfixed in formalin and processed by routine techniques. Bone marrowbiopsies and aspirate sections were obtained and processed by routinetechniques.

Determination of Immunoglobulin Mutational Status.

Five hundred nanograms of mRNA or 1-5 μg of total RNA was used togenerated oligo-dT primed cDNA using Superscript (Life Technologies).Amplification of the immunoglobulin V-heavy sequence was performedessentially as described (Hamblin et al., Blood 94:1848, 1999; Fais etal., J. Clin. Invest., 102:1515, 1998; Campbell et al., Mol. Immunol.,29:193, 1992). In brief: cDNA was amplified by polymerase chain reaction(PCR) using a mixture of 5′ oligonucleotides specific for each leadersequence of the VH1 to VH7 IgVH families as forward primers (VH1 andVH7: 5′-CCA TGG ACT GGA CCT GGA-3′ (SEQ ID NO: 5); VH2: 5′-ATG GAC ATACTT TGT TCC AC-3′ (SEQ ID NO: 6); VH3: 5′-CCA TGG AGT TTG GGC TGA GC-3′(SEQ ID NO: 7); VH4: 5′-ATG AAA CAC CTG TGG TTC TT-3′ (SEQ ID NO: 8);VH5: 5′-ATG GGG TCA ACC GCC ATC CT-3′ (SEQ ID NO: 9); VH6: 5′-ATG TCTGTC TCC TTC CTC AT-3′) (SEQ ID NO: 10)) and either a 3′ oligonucleotidecomplementary to the JH consensus sequence (5′-ACC TGA GGA GAC GGT GACC-3′; SEQ ID NO: 11) or the constant region of the IgM locus (5′-AGG AGAAAG TGA TGG AGT CG-3; SEQ ID NO: 12) as reverse primers (Campbell etal., Mol. Immunol. 29:193, 1992; Fais et al., J. Clin. Invest. 102:1515,1998). For samples that failed to amplify with this combination, IgVHfamily specific primers complementary to the 5′ end of framework region(FR)1 were used (FR1-VH1: 5′-AGG TGC AGC TGG TGC AGT CTG-3′ (SEQ ID NO:16); FR1-VH2: 5′-AGG TCA ACT TAA GGG AGT CTG (SEQ ID NO: 17); FR1-VH3:5′-AGG TGC AGC TGG TGG AGT CTG-3′ (SEQ ID NO: 18); FR1-VH4: 5′-AGG TGCAGC TGC AGG AGT CGG-3′ (SEQ ID NO: 19); FR1-VH5: 5′-AGG TGC AGC TGC TGCAGT CTG-3′ (SEQ ID NO: 20); FR1-VH6: 5′-AGG TAC AGC TGC AGC AGT CAG-3′(SEQ ID NO: 21); (Marks et al., Eur. J. Immunol. 21:985, 1991)).

PCR was performed in 50 μL reactions with Taq polymerase (Sigma) and 20pmol of each primer. Cycling conditions were 94° C. 30 sec, 60° C. 20sec, 72° C. 30 sec for up to 35 cycles. Products were purified (MinElutePCR Purification Kit, Qiagen) and sequenced directly with theappropriate 3′ oligonucleotide using Big Dye Terminator and analyzed onan automated DNA sequencer (Applied Biosystems). Nucleotide sequenceswere aligned to the V-Base sequence directory found on the MedicalResearch Council Centre for Protein Engineering website. Percentagehomology was calculated by counting the number of mutations between the5′ end of FR1 and the 3′ end of FR3. Sequences with <2% deviation fromthe germline VH sequence were considered unmutated (Hamblin et al.,Blood 94:1848, 1999).

CD38 Expression Analysis.

Whole blood was stained within 24 hours of collection with a panel ofantibodies as previously described (Fukushima et al., Cytometry, 26:243,1996). Five-parameter, three-color flow cytometry was performed with aFACS Calibur flow cytometer and analyzed with CellQuest software(BectonDickinson). Lymphocytes were gated by forward and side scatter.Isotype controls were run with each patient specimen. CD38 positivecells were determined as the percent of lymphocytes staining moreintensely with anti-CD38 (CD38-PE, Becton Dickinson) than with isotypecontrol.

DNA Microarray Analysis.

The DNA microarray methods have been described in detail in Example 1.Fluorescently labeled cDNA probes were generated from mRNA (Fast Track,Invitrogen), using the Cy5 dye to label cDNA from the CLL samples, andthe Cy3 dye to label cDNA from a reference pool of mRNA prepared from 9lymphoma cell lines (Alizadeh et al., Nature 403:503, 2000). LymphochipDNA microarrays containing 13,868 human cDNAs were prepared and used aspreviously described (see Example 1, Alizadeh et al., Nature 403:503,2000). Initial microarray data selection was based on fluorescencesignal intensity, with the requirement of 50 relative fluorescent units(RFU's) above background in both the Cy3 and Cy5 channels, or 500 RFU'sabove background in either channel alone.

Protein Lysates and Western Blotting.

Twenty million CD19+ purified CLL cells were lysed in 1 mL of lysisbuffer containing 1% Triton. Protein concentration was determined byBradford assay. 12 μg of protein per lane was loaded on precast SDS gels(Invitrogen) and separated and transferred to nitrocellulose asrecommended by the manufacturer. Western blots were incubated with amouse monoclonal antibody to ZAP-70 (clone 2F3.2, Upstate Biotechnology)in PBS with 4% milk. Secondary staining was done with horseradishperoxidase coupled anti mouse antibodies and chemiluminescence(Amersham).

Immunohistochemistry

Immunohistochemistry was performed on deparaffinized sections, takenfrom neutral buffered formalin-fixed, paraffin-embedded (FFPE) tissueusing a panel of monoclonal and polyclonal antibodies (listed below).Bone marrow trephine biopsies were also decalcified prior to sectioning.In brief, the deparaffinized slides were placed in a microwaveablepressure cooker containing 1.5 liters of 10 mM citrate buffer (pH of6.0) containing 0.1% Tween 20, and microwaved (Model R4A80, SharpElectronics, Rahwah, N.J.) for 40 min at 700 watts. Antigens werelocalized using an avidin-biotin-peroxidase method with3,3′-diaminobenzidine as a chromogen and performed using an automatedimmunostainer (Ventana Medical Systems, Inc., Tucson, Ariz.) accordingto the manufacturer's protocol with minor modifications. Primaryantibody incubation was performed for 2 hours. Positive and negativecontrols were run with all cases and stained appropriately. Anti-CD3 andanti-CD20 antibodies were obtained from Dako (Carpenteria, Calif.). Anindependent pathologist scored all slides in a blinded fashion.

TABLE 1 Antibodies and conditions used for immunohistochemistry. AntigenClone Dilution Source ZAP-70 2F3.2 1:80 Upstate Biotechnology (LakePlacid, NY) ZAP-70 Polyclonal (sc-574) 1:20 Santa Cruz Biotechnology(Santa Cruz, CA) ZAP-70 mouse monoclonal 1:100 BD TransductionLaboratories (Lexington, KY) CD3 Polyclonal 1:100 Dako (Carpinteria, CA)CD5 4C7 1:50 Novocastra (Newcastle Upon Tyne, England) CD20 L26 1:200Dako

Flow Cytometry.

Cell lines or ficolled PBMCs were fixed and permeabilized usingcommercially available kits (Fix and Perm, Caltag; Intrastain, Dako)stained with 0.2-1 μg of primary antibody against ZAP-70 (UpstateBiotechnology) and PE labeled secondary rat anti mouse antibody (BectonDickinson). Cells were analyzed on a FACSort (Becton Dickinson).

Statistical Analysis.

A two-group t-statistic on log2-transformed mRNA expression ratios wasused to measure the ability of each array element to discriminatebetween the two IgVH mutation subtypes of CLL univariately. To create atest for this subtype distinction based on ZAP-70 mRNA expression, thepatients were divided into two groups based on a cut point of ZAP-70expression that minimized the classification errors. Time to treatmentmeasured from diagnosis was estimated by the Kaplan-Meier method andcompared by the log-rank test.

Quantitative RT-PCR.

An aliquot of the same mRNA used for the DNA microarray study wasdiluted to approximately 0.5 ng/μL. Five μL of the diluted mRNA perreaction was used for quantitative RT-PCR using TaqMan™ reagents andanalyzed in real time on an ABI Prism 7700 Sequence Detector asrecommended by the manufacturer (Applied Biosystems). All samples wererun in triplicates. Amplification of the sequence of interest wascompared to a reference probe (β-2-microglobulin) and normalized againsta standard curve of Jurkat cell mRNA. Primers and probes forβ-2-microglobulin and Cyclin D1 have been described (Bijwaard et al.,Clin. Chem. 47:195, 2001) and for ZAP-70 these were5′-CGCTGCACAAGTTCCTGGT-3′ (forward primer, SEQ ID NO: 13),5′-GACACCTGGTGCAGCAGCT-3′ (reverse primer, SEQ ID NO: 14) and5′-CATTGCTCACAGGGATCTCCTCCCTCT-3′ (FAM™-probe, SEQ ID NO: 15).

Results CLL Subtype Distinction Genes.

In this study, Lymphochip DNA microarrays were used to profile geneexpression in CD19⁺ purified CLL samples from a cohort of 39 patients inorder to identify the genes that most accurately discriminate betweenthe CLL subtypes, which could potentially be used in a clinical test forthis distinction. Using a conventional cutoff of 98% sequence identityto the nearest germline IgVH sequence, 28 cases (72%) were classified asIg-mutated CLL and 11 cases (28%) were classified as Ig-unmutated CLL.Of the Ig-unmutated CLL samples, seven (64%) were 100% identical to agermline IgVH sequence and four (36%) were 98-99% identical. TheIg-unmutated and Ig-mutated CLL samples differentially expressed ˜240genes (304 microarray elements) with high statistical significance(p<0.001). These differentially expressed genes included many that wereidentified in pilot gene expression profiling studies of CLL (seeExample 1, above; Klein et al., J. Exp. Med. 194:1625, 2001). ZAP-70 wasby far the best subgroup distinction gene in the present analysis.ZAP-70 expression was, on average, 5.54 fold higher in Ig-unmutated CLLthan in Ig-mutated CLL and distinguished the subtypes with a statisticalsignificance of p<10⁻²¹.

ZAP-70 and CD38 as Surrogate Markers of IgVH Mutation Status.

IgVH mutation status confers important prognostic information, but IgVHsequencing is not suitable for most clinical laboratories. It wastherefore investigated whether ZAP-70 mRNA expression could be used as asurrogate marker for this distinction. To do this, a cut point wasdetermined based on ZAP-70 expression levels that would optimallydistinguish most Ig-unmutated CLL samples from most Ig-mutated CLLsamples. Using this cut point, 95% of the CLL samples could beclassified into the correct CLL subtype (FIG. 2). Two samples (5%) werediscordant for IgVH mutation status and ZAP-70 expression. Among the 281g-mutated CLL samples, two showed ZAP-70 expression levels comparable toIg-unmutated CLL. Other genes, were searched for that had expressionpatterns that could be combined with ZAP-70 expression to create amultivariate classifier that would perform better than ZAP-70 expressionalone. No such gene was found.

CD38 surface expression has been shown to be a surrogate marker of IgVHmutation status in some studies (Damle et al., Blood, 94:1840, 1999).Flow cytometric analysis of CD38 expression was available on 36 patientsin this study. Early studies considered CLL cases with >30%CD38-expressing cells to be CD38⁺ (Damle et al., Blood, 99:4087, 2002).Based on this criterion, 11 (31%) of our CLL samples were CD38⁺. Morerecently, CD38⁺ CLL cases were defined as those with >7% CD38-expressingcells, based on overall survival analysis in 200 patients (Krober etal., Blood, 100:1410, 2002). In the present series, 14 patients (39%)were CD38+ by this criterion. As expected, CD38 expression tended to behigher in Ig-unmutated CLL samples, but there was a considerable overlapin CD38 expression between the CLL subtypes. Overall, CD38 predictedIgVH mutation status correctly in 86% of patients when a cutoff of 30%was used and in 78% when a cutoff of 7% was applied. Thus, considerablymore patients were misclassified by CD38 expression than by ZAP-70expression. Furthermore, CD38 expression yielded more false positive(27%) and false negative (8%) assignments than did ZAP-70 expression(15% and 0%, respectively).

Several groups have reported the prognostic value of CD38 expressionwithout correlating it with IgVH mutational status (Hamblin et al., Ann.Hematol., 81:299, 2002; D'Arena et al., Leuk. Lymphoma, 42:109, 2001;Del Poeta et al., Blood, 98:2633, 2001′ Durig et al., Leukemia, 16:30,2002; Ibrahim et al., Blood, 98:181, 2001; Heintel et al., Leuk.Lymphoma, 42:1315, 2001; Morabito et al., Leuk. Res. 25:927, 2001). IgVHmutation status, ZAP-70 mRNA expression and CD38 surface expression werecompared for their ability to predict time to disease progression, asjudged by treatment requirement. At the time of last follow-up, tenpatients (26%) had been treated. The CLL patients were divided into twogroups based on the ZAP-70 expression cutoff described above, and thesegroups differed significantly in their time to treatment followingdiagnosis (p=0.01). ZAP-70 expression and IgVH mutation status werecomparable in their ability to define CLL patients, who were differentwith respect to disease progression (FIGS. 3A, 3B).

Patients Discordant for IgVH Mutational Status and ZAP-70 Expressionhave Distinct Biological and Clinical Characteristics.

As mentioned above, 2 patients were discordant for ZAP-70 expression andIgVH mutation status, and these patients will be referred to as ZAP-70outliers. All ZAP-70 outliers fulfilled the diagnostic criteria for CLL,and the cytogenetic abnormalities in the leukemic cells of thesepatients were typical of CLL (Table 2).

TABLE 2 Characteristics of patients discordant for mutational status andZAP-70 expression. Months CLL Sex/ IgVH ZAP-70 CD 38 Cyto- Clinical to #Age IgVH gene mRNA positive genetics course therapy M2 F/54 96% 3-21 (+)83% 13q- stable n.e. M4 M/82 95% 3-21 (+) 18% normal treated 20 IgVH: %homology to germline IgVH gene; ZAP-70 mRNA expression as determined bymicroarray analysis; % CD38 positive (i.e. staining above isotypecontrol) by flow cytometry; +12: trisomy 12; 13q-: 13q deletion; 11q-:11q deletion; n.e.: not evaluable.

Intriguingly, the CLL cells of the two outlier patients expressed amutated VH3-21 gene, and these were the only cases in this series thatutilized this IgVH gene. This finding is notable since expression of amutated VH3-21 gene has been associated with progressive disease and mayrepresent a biologically distinct subset of Ig-mutated CLL.

ZAP-70 Assays for Potential Clinical Application

The findings outlined above demonstrate that ZAP-70 mRNA expression, asmeasured by DNA microarrays, can be used to assign the majority of CLLpatients to the correct IgVH mutational subtype and can identify patientsubsets that have distinct treatment requirements. A clinical test basedon ZAP-70 expression would therefore be a useful adjunct in patientmanagement.

To this end, a quantitative RT-PCR assay for ZAP-70 expression wasdevised, and the quantitation of ZAP-70 mRNA levels by this method wascompared with the results from the DNA microarray analysis (FIG. 4). Thetwo assays showed an excellent correlation over a wide range of ZAP-70mRNA levels. These results confirm the quantitative nature of the DNAmicroarray measurements, and suggest that a quantitative RT-PCR assaymight be suitable for measuring ZAP-70 expression in a clinical setting.However, this assay requires that the leukemic cells be purified, sinceZAP-70 is highly expressed in T cells.

The possibility that ZAP-70 protein levels could be used for a clinicaldiagnostic test was also demonstrated. Western blots of lysates of CD19⁺CLL cells revealed high levels of ZAP-70 protein expression inIg-unmutated CLL samples compared with the relatively low levels inIg-mutated CLL samples (FIG. 5A). Since this assay quantitates ZAP-70protein in a population of cells, T cells were removed given their highZAP-70 protein expression.

To demonstrate that ZAP-70 protein expression is detectable byimmunohistochemistry, peripheral blood and routine bone marrow biopsysamples from CLL patients (FIG. 5B) were studied. In all samples, Tcells stained strongly for ZAP-70, as expected. In samples fromIg-mutated CLL patients, the leukemic cells were negative or weak forZAP-70 staining whereas the interspersed T cells were strongly positive.In samples from Ig-unmutated CLL, both the leukemic cells and the Tcells had readily detectable staining.

ZAP-70 expression in 100 clinical samples from 43 patients was analyzed.In a blinded analysis, a pathologist assigned 37 of the 43 patients(86%) correctly to the IgVH mutation subtype based on ZAP-70 staining byimmunohistochemistry. Two misclassified patients were ZAP-70 outliers inthe DNA microarray analysis and used the mutated VH3-21 IgVH gene. Thus,in these patients the immunohistochemistry was in accord with the mRNAmeasurement. Interestingly, in two further misclassified cases, ZAP-70was positive only in a subset of the leukemic cells comprising less than25% of the sample. This finding indicates clonal heterogeneity in thesepatients and could be of biologic significance. The two remainingmisclassified cases were low for ZAP-70 mRNA expression and thereforewere false positive by immunohistochemistry.

ZAP-70 Expression by Flow Cytometry.

Flow cytometry for surface markers is widely used in the diagnosis ofCLL/SLL. A T cell line, which expresses ZAP-70, and a B-cell line, whichis negative for ZAP-70, were used to establish flow cytometry conditionsfor ZAP-70, which due to its intracellular location is more difficult todetect. Using clinical samples the detection of ZAP-70 is currently lessreliable. Optimization of fixation and permeabilization conditions andfluorescence coupled antibodies against ZAP-70 will increase thereliability of this assay and could be combined in a diagnostic kit.Similarly, some clinical laboratories might prefer immunocytochemistry,to which the same considerations apply.

ZAP-70 Protein Expression is a Clinically Useful Prognostic Marker andCorrelates with IgVH Gene Mutation Status.

It is disclosed herein that ZAP-70 mRNA expression is an excellentsurrogate marker for the distinction between the Ig-mutated andIg-unmutated CLL subtypes and can identify patient groups with divergentclinical courses. ZAP-70 expression assigned 93% of the patients studiedto the correct Ig mutation subtype. No other gene represented on themicroarrays was as good as ZAP-70 in making this CLL subtypedistinction, nor could any other gene improve the predictive power ofZAP-70. High ZAP-70 expression identified a clinically progressive formof CLL. By contrast, patients whose leukemic cells had low ZAP-70expression had an indolent disease.

Hence ZAP-70 expression is believed to be a useful clinical test toguide treatment decisions. The measurement of ZAP-70 expression had arelatively low false positive rate compared to CD38. Early treatment maybe beneficial for patients whose CLL cells have high ZAP-70 expression.By contrast, patients whose CLL cells have low ZAP-70 expression may bemanaged best by delaying treatment for as long as possible. Otherprognostic markers such as chromosomal abnormalities (i.e. 11q or 17pdeletion) can also be taken into consideration when designing protocolsfor stratifying the treatment of CLL patients.

ZAP-70 expression can be evaluated in a clinical diagnostic laboratoryusing a variety of approaches. A strong correlation between the ZAP-70mRNA levels measured by DNA microarray and by quantitative RT-PCR wasdemonstrated (see Examples 1 and 2, above). Quantitative PCR assays haveexceedingly low variation in measurement, and thus are ideal foraccurately discriminating the CLL subtypes based on ZAP-70 expression.It was also demonstrated that ZAP-70 protein expression, as detected byan immunohistochemical assay, correlated well with Ig mutational status.A protein expression assay could be readily performed withoutpurification of the CLL cells.

Thus, testing for ZAP-70 expression is an easily performed clinicalassay to distinguish prognostic groups of CLL/SLL. Compared to RNA/DNAbased techniques or western blots, the use of immunohistochemistry, flowcytometry, or immunofluorescence has several advantages. First, thesetechniques are in routine use in clinical laboratories. They do notrequire extensive purification of cells prior to analysis and severalproteins of interest can be analyzed concomitantly. These tests lendthemselves well to the development of diagnostic kits that will help tomake the test widely available and will increase reliable performance ofthe assays. An alternative, complementary approach is quantitative ELISAto measure the total amount of ZAP-70 in a blood sample.

Example 3 Methods of Making Human ZAP-70 cDNA

The following example provides representative techniques for preparingcDNA.

Total RNA is extracted from human cells by any one of a variety ofmethods well known to those of ordinary skill in the art. Sambrook etal. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989)and Ausubel et al. (In Current Protocols in Molecular Biology, GreenePubl. Assoc. and Wiley-Intersciences, 1992) provide descriptions ofmethods for RNA isolation. The extracted RNA is then used as a templatefor performing reverse transcription-polymerase chain reaction (RT-PCR)amplification of cDNA. Methods and conditions for RT-PCR are described,for instance, in Kawasaki et al., (In PCR Protocols, A Guide to Methodsand Applications, Innis et al. (eds.), 21-27, Academic Press, Inc., SanDiego, Calif., 1990).

The selection of amplification primers is made according to theportion(s) of the cDNA that is to be amplified. Primers may be chosen toamplify a segment of a cDNA or the entire cDNA molecule. Variations inamplification conditions may be required to accommodate primers andamplicons of differing lengths and composition; such considerations arewell known in the art and are discussed for instance in Innis et al.(PCR Protocols, A Guide to Methods and Applications, Academic Press,Inc., San Diego, Calif., 1990). By way of example, a human ZAP-70 cDNAmolecule is amplified using the primers and conditions described inExample 1.

Those primers and conditions are illustrative only; one skilled in theart will appreciate that many different primers may be derived from theprovided cDNA sequence in order to amplify particular regions of ZAP-70cDNA, as well as the complete sequence of the human ZAP-70 cDNA.

Re-sequencing of PCR products obtained by these amplification proceduresis advantageous to facilitate confirmation of the amplified sequence andprovide information about natural variation of this sequence indifferent populations or species. Oligonucleotides derived from theprovided ZAP-70 sequences may be used in such sequencing methods.

Orthologs of human ZAP-70 can be cloned in a similar manner, where thestarting material consists of cells taken from a non-human species.Orthologs will generally share at least 65% sequence identity with thedisclosed human ZAP-70 cDNA. Where the non-human species is more closelyrelated to humans, the sequence identity will in general be greater.Closely related orthologous ZAP-70 molecules may share at least 70%, atleast 75%, at least 80% at least 85%, at least 90%, at least 91%, atleast 93%, at least 95%, or at least 98% sequence identity with thedisclosed human sequences.

Oligonucleotides derived from the human ZAP-70 cDNA sequence, orfragments of this cDNA, are encompassed within the scope of the presentdisclosure. Such oligonucleotides may comprise a sequence of at least 15consecutive nucleotides of the ZAP-70 nucleic acid sequence. If theseoligonucleotides are used with an in vitro amplification procedure (suchas PCR), lengthening the oligonucleotides may enhance amplificationspecificity. Thus, oligonucleotide primers comprising at least 25, 30,35, 40, 45 or 50 consecutive nucleotides of these sequences may be used.These primers for instance may be obtained from any region of thedisclosed sequences. By way of example, the human ZAP-70 cDNA, ORF andgene sequences may be apportioned into about halves or quarters based onsequence length, and the isolated nucleic acid molecules (e.g.,oligonucleotides) may be derived from the first or second halves of themolecules, or any of the four quarters

Nucleic acid molecules may be selected that comprise at least 15, 20,23, 25, 30, 35, 40, 50 or 100 consecutive nucleotides of any of these orother portions of the human ZAP-70 cDNA. Thus, representative nucleicacid molecules might comprise at least 15 consecutive nucleotides of thehuman ZAP-70 cDNA (SEQ ID NO: 1).

Example 4 Expression of ZAP-70 Protein

The expression and purification of the ZAP-70 protein, and fragmentsthereof, are carried out using standard laboratory techniques. Purifiedhuman ZAP-70 protein (or fragments thereof) may be used for functionalanalyses, drug development, testing and analysis, antibody production,diagnostics, and patient therapy. Furthermore, the DNA sequence of theZAP-70 cDNA can be manipulated in studies to understand the expressionof the gene and the function of its product. Mutant forms of the humanZAP-70 may be isolated based upon information contained herein, and maybe studied in order to detect alteration in expression patterns in termsof relative quantities, cellular localization, tissue specificity andfunctional properties of the encoded mutant ZAP-70 protein.

Partial or full-length cDNA sequences, which encode for the subjectprotein, may be ligated into bacterial expression vectors. Methods forexpressing large amounts of protein from a cloned gene introduced intoEscherichia coli (E. coli) may be utilized for the purification,localization and functional analysis of proteins. For example, fusionproteins consisting of amino terminal peptides encoded by a portion ofthe E. coli lacZ or trpE gene linked to ZAP-70 proteins may be used toprepare polyclonal and monoclonal antibodies against these proteins.Thereafter, these antibodies may be used to purify proteins byimmunoaffinity chromatography, in diagnostic assays to quantitate thelevels of protein and to localize proteins in tissues and individualcells by immunofluorescence. Such antibodies may be specific for epitopetags, which can be added to the expression construct for identificationan/or purification purposes.

Intact native protein may also be produced in E. coli in large amountsfor functional studies. Methods and plasmid vectors for producing fusionproteins and intact native proteins in bacteria are described inSambrook et al. (Sambrook et al., In Molecular Cloning: A LaboratoryManual, Ch. 17, CSHL, New York, 1989). Such fusion proteins may be madein large amounts, are easy to purify, and can be used to elicit antibodyresponse. Native proteins can be produced in bacteria by placing astrong, regulated promoter and an efficient ribosome-binding siteupstream of the cloned gene. If low levels of protein are produced,additional steps may be taken to increase protein production; if highlevels of protein are produced, purification is relatively easy.Suitable methods are presented in Sambrook et al. (In Molecular Cloning:A Laboratory Manual, CSHL, New York, 1989) and are well known in theart. Often, proteins expressed at high levels are found in insolubleinclusion bodies. Methods for extracting proteins from these aggregatesare described by Sambrook et al. (In Molecular Cloning: A LaboratoryManual, Ch. 17, CSHL, New York, 1989). Vector systems suitable for theexpression of lacZ fusion genes include the pUR series of vectors(Ruther and Muller-Hill, EMBO J. 2:1791, 1983), pEX1-3 (Stanley andLuzio, EMBO J. 3:1429, 1984) and pMR100 (Gray et al., Proc. Natl. Acad.Sci. USA 79:6598, 1982). Vectors suitable for the production of intactnative proteins include pKC30 (Shimatake and Rosenberg, Nature 292:128,1981), pKK177-3 (Amann and Brosius, Gene 40:183, 1985) and pET-3(Studiar and Moffatt, J. Mol. Biol. 189:113, 1986). ZAP-70 fusionproteins may be isolated from protein gels, lyophilized, ground into apowder and used as an antigen. The DNA sequence can also be transferredfrom its existing context to other cloning vehicles, such as otherplasmids, bacteriophages, cosmids, animal viruses and yeast artificialchromosomes (YACs) (Burke et al., Science 236:806-812, 1987). Thesevectors may then be introduced into a variety of hosts including somaticcells, and simple or complex organisms, such as bacteria, fungi(Timberlake and Marshall, Science 244:1313-1317, 1989), invertebrates,plants, and animals (Pursel et al., Science 244:1281-1288, 1989), whichcells or organisms are rendered transgenic by the introduction of theheterologous ZAP-70 cDNA.

For expression in mammalian cells, the cDNA sequence may be ligated toheterologous promoters, such as the simian virus (SV) 40 promoter in thepSV2 vector (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076,1981), and introduced into cells, such as monkey COS-1 cells (Gluzman,Cell 23:175-182, 1981), to achieve transient or long-term expression.The stable integration of the chimeric gene construct may be maintainedin mammalian cells by biochemical selection, such as neomycin (Southernand Berg, J. Mol. Appl. Genet. 1:327-341, 1982) and mycophenolic acid(Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981).

DNA sequences can be manipulated with standard procedures such asrestriction enzyme digestion, fill-in with DNA polymerase, deletion byexonuclease, extension by terminal deoxynucleotide transferase, ligationof synthetic or cloned DNA sequences, site-directed sequence-alterationvia single-stranded bacteriophage intermediate or with the use ofspecific oligonucleotides in combination with nucleic acidamplification.

The cDNA sequence (or portions derived from it) or a mini gene (a cDNAwith an intron and its own promoter) may be introduced into eukaryoticexpression vectors by conventional techniques. These vectors aredesigned to permit the transcription of the cDNA in eukaryotic cells byproviding regulatory sequences that initiate and enhance thetranscription of the cDNA and ensure its proper splicing andpolyadenylation. Vectors containing the promoter and enhancer regions ofthe SV40 or long terminal repeat (LTR) of the Rous Sarcoma virus andpolyadenylation and splicing signal from SV40 are readily available(Mulligan et al., Proc. Natl. Acad. Sci. USA 78:1078-2076, 1981; Gormanet al., Proc. Natl. Acad. Sci. USA 78:6777-6781, 1982). The level ofexpression of the cDNA can be manipulated with this type of vector,either by using promoters that have different activities (for example,the baculovirus pAC373 can express cDNAs at high levels in S. frugiperdacells (Summers and Smith, In Genetically Altered Viruses and theEnvironment, Fields et al. (Eds.) 22:319-328, CSHL Press, Cold SpringHarbor, N.Y., 1985) or by using vectors that contain promoters amenableto modulation, for example, the glucocorticoid-responsive promoter fromthe mouse mammary tumor virus (Lee et al., Nature 294:228, 1982). Theexpression of the cDNA can be monitored in the recipient cells 24 to 72hours after introduction (transient expression).

In addition, some vectors contain selectable markers such as the gpt(Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981) orneo (Southern and Berg, J. Mol. Appl. Genet. 1:327-341, 1982) bacterialgenes. These selectable markers permit selection of transfected cellsthat exhibit stable, long-term expression of the vectors (and thereforethe cDNA). The vectors can be maintained in the cells as episomal,freely replicating entities by using regulatory elements of viruses,such as papilloma (Sarver et al., Mol. Cell. Biol. 1:486-496, 1981) orEpstein-Barr (Sugden et al., Mol. Cell. Biol. 5:410-413, 1985).Alternatively, one can also produce cell lines that have integrated thevector into genomic DNA. Both of these types of cell lines produce thegene product on a continuous basis. One can also produce cell lines canalso produced that have amplified the number of copies of the vector(and therefore of the cDNA as well) to create cell lines that canproduce high levels of the gene product (Alt et al., J. Biol. Chem.253:1357-1370, 1978).

The transfer of DNA into eukaryotic, in particular human or othermammalian cells, is now a conventional technique. Recombinant expressionvectors can be introduced into the recipient cells as pure DNA(transfection) by, for example, precipitation with calcium phosphate(Graham and vander Eb, Virology 52:466, 1973) or strontium phosphate(Brash et al., Mol. Cell. Biol. 7:2013, 1987), electroporation (Neumannet al., EMBO J. 1:841, 1982), lipofection (Feigner et al., Proc. Natl.Acad. Sci. USA 84:7413, 1987), DEAE dextran (McCuthan et al., J. Natl.Cancer Inst. 41:351, 1968), microinjection (Mueller et al., Cell 15:579,1978), protoplast fusion (Schafner, Proc. Natl. Acad. Sci. USA77:2163-2167, 1980), or pellet guns (Klein et al., Nature 327:70, 1987).Alternatively, the cDNA, or fragments thereof, can be introduced byinfection with virus vectors. Systems are developed that use, forexample, retroviruses (Bernstein et al., Gen. Engr'g 7:235, 1985),adenoviruses (Ahmad et al., J. Virol. 57:267, 1986), or Herpes virus(Spaete et al., Cell 30:295, 1982). Techniques of use in packaging longtranscripts can be found in Kochanek et al. (Proc. Natl. Acad. Sci. USA93:5731-5739, 1996) Parks et al. (Proc. Natl. Acad. Sci. USA93:13565-13570, 1996) and Parks and Graham (J. Virol. 71:3293-3298,1997). ZAP-70 encoding sequences can also be delivered to target cellsin vitro via non-infectious systems, for instance liposomes.

These eukaryotic expression systems can be used for studies of ZAP-70encoding nucleic acids and mutant forms of these molecules, the ZAP-70protein and mutant forms of this protein. Such uses include, forexample, the identification of regulatory elements located in the 5′region of the ZAP-70 gene on genomic clones that can be isolated fromhuman genomic DNA libraries using the information contained herein. Theeukaryotic expression systems also may be used to study the function ofthe normal complete protein, specific portions of the protein, or ofnaturally occurring or artificially produced mutant proteins, and inorder to analyze and characterize inhibitory molecules that can be usedto reduce the activity of ZAP-70 in vitro of in vivo.

Using the above techniques, expression vectors containing the ZAP-70gene sequence or cDNA, or fragments or variants or mutants thereof, canbe introduced into human cells, mammalian cells from other species ornon-mammalian cells, as desired. The choice of cell is determined by thepurpose of the treatment. For example, monkey COS cells (Gluzman, Cell23:175-82, 1981) that produce high levels of the SV40 T antigen andpermit the replication of vectors containing the SV40 origin ofreplication may be used. Similarly, Chinese hamster ovary (CHO), mouseNIH 3T3 fibroblasts or human fibroblasts or lymphoblasts may be used.

Embodiments described herein thus encompass recombinant vectors thatcomprise all or part of a ZAP-70 encoding sequence, such as the ZAP-70gene or cDNA or variants thereof, for expression in a suitable host. TheZAP-70 DNA is operatively linked in the vector to an expression controlsequence in the recombinant DNA molecule so that the ZAP-70 polypeptidecan be expressed. The expression control sequence may be selected fromthe group consisting of sequences that control the expression of genesof prokaryotic or eukaryotic cells and their viruses and combinationsthereof. The expression control sequence may be specifically selectedfrom the group consisting of the lac system, the trp system, the tacsystem, the trc system, major operator and promoter regions of phagelambda, the control region of fd coat protein, the early and latepromoters of SV40, promoters derived from polyoma, adenovirus,retrovirus, baculovirus and simian virus, the promoter for3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, thepromoter of the yeast alpha-mating factors and combinations thereof.

The host cell, which may be transfected with a vector, may be selectedfrom the group consisting of E. coli, Pseudomonas, Bacillus subtilis,Bacillus stearothermophilus or other bacilli; other bacteria; yeast;fungi; insect; mouse or other animal; or plant hosts; or human tissuecells.

It is appreciated that for mutant or variant ZAP-70 DNA sequences,similar systems are employed to express and produce the mutant product.

Example 5 Production of an Antibody to ZAP-70 Protein or ProteinFragments

Monoclonal or polyclonal antibodies may be produced to either the normalZAP-70 protein or mutant forms of this protein. Optimally, antibodiesraised against the ZAP-70 protein would specifically detect the ZAP-70protein. That is, such antibodies would recognize and bind the ZAP-70protein and would not substantially recognize or bind to other proteinsfound in human cells. Antibodies the human ZAP-70 protein may recognizeZAP-70 from other species, such as murine ZAP-70, and vice versa.

The determination that an antibody specifically detects the ZAP-70protein is made by any one of a number of standard immunoassay methods;for instance, the Western blotting technique (Sambrook et al., InMolecular Cloning: A Laboratory Manual, CSHL, New York, 1989). Todetermine that a given antibody preparation (such as one produced in amouse) specifically detects the ZAP-70 protein by Western blotting,total cellular protein is extracted from human cells (for example,lymphocytes) and electrophoresed on a sodium dodecylsulfate-polyacrylamide gel. The proteins are then transferred to amembrane (for example, nitrocellulose or PVDF) by Western blotting, andthe antibody preparation is incubated with the membrane. After washingthe membrane to remove non-specifically bound antibodies, the presenceof specifically bound antibodies is detected by the use of (by way ofexample) an anti-mouse antibody conjugated to an enzyme such as alkalinephosphatase. Application of an alkaline phosphatase substrate5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium results inthe production of a dense blue compound by immunolocalized alkalinephosphatase. Antibodies that specifically detect the ZAP-70 proteinwill, by this technique, be shown to bind to the ZAP-70 protein band(which will be localized at a given position on the gel determined byits molecular weight, which is approximately 125 kDa based ongel-mobility estimation for murine ZAP-70. Non-specific binding of theantibody to other proteins may occur and may be detectable as a weaksignal on the Western blot. The non-specific nature of this binding willbe recognized by one skilled in the art by the weak signal obtained onthe Western blot relative to the strong primary signal arising from thespecific antibody- ZAP-70 protein binding.

Substantially pure ZAP-70 protein suitable for use as an immunogen canbe isolated from the transfected or transformed cells as describedabove. The concentration of protein in the final preparation isadjusted, for example, by concentration on an Amicon (Millipore,Bedford, Mass.) or similar filter device, to the level of a fewmicrograms per milliliter. Monoclonal or polyclonal antibody to theprotein can then be prepared as follows:

A. Monoclonal Antibody Production by Hybridoma Fusion

Monoclonal antibody to epitopes of the ZAP-70 protein identified andisolated as described can be prepared from murine hybridomas accordingto the classical method of Kohler and Milstein (Nature 256:495-497,1975) or derivative methods thereof. Briefly, a mouse is repetitivelyinoculated with a few micrograms of the selected protein over a periodof a few weeks. The mouse is then sacrificed, and the antibody-producingcells of the spleen isolated. The spleen cells are fused with mousemyeloma cells using polyethylene glycol, and the excess un-fused cellsdestroyed by growth of the system on selective media comprisingaminopterin (HAT media). Successfully fused cells are diluted andaliquots of the dilution placed in wells of a microtiter plate, wheregrowth of the culture is continued. Antibody-producing clones areidentified by detection of antibody in the supernatant fluid of thewells by immunoassay procedures, such as ELISA, as originally describedby Engvall (Enzymol. 70(A):419-439, 1980), and derivative methodsthereof. Selected positive clones can be expanded and their monoclonalantibody product harvested for use. Detailed procedures for monoclonalantibody production are described in Harlow and Lane (Antibodies, ALaboratory Manual, CSHL, New York, 1988).

B. Polyclonal Antibody Production by Immunization

Polyclonal antiserum containing antibodies to heterogeneous epitopes ofa single protein can be prepared by immunizing suitable animals with theexpressed protein (Example 4), which optionally can be modified toenhance immunogenicity. Effective polyclonal antibody production isaffected by many factors related both to the antigen and the hostspecies. For example, small molecules tend to be less immunogenic thanothers and may require the use of carriers and adjuvant, examples ofwhich are known. Also, host animals vary in response to site ofinoculations and dose, with either inadequate or excessive doses ofantigen resulting in low titer antisera. A series of small doses (nglevel) of antigen administered at multiple intradermal sites appear tobe most reliable. An effective immunization protocol for rabbits can befound in Vaitukaitis et al. (J. Clin. Endocrinol. Metab. 33:988-991,1971).

Booster injections can be given at regular intervals, and antiserumharvested when antibody titer thereof begins to fall, as determinedsemi-quantitatively (for example, by double immunodiffusion in agaragainst known concentrations of the antigen). See, for example,Ouchterlony et al. (In Handbook of Experimental Immunology, Wier, D.(ed.) chapter 19. Blackwell, 1973). Plateau concentration of antibody isusually in the range of about 0.1 to 0.2 mg/ml of serum (about 12 μM).Affinity of the antisera for the antigen is determined by preparingcompetitive binding curves, as described, for example, by Fisher (Manualof Clinical Immunology, Ch. 42, 1980).

C. Antibodies Raised Against Synthetic Peptides

A third approach to raising antibodies against the ZAP-70 protein is touse synthetic peptides synthesized on a commercially available peptidesynthesizer based upon the predicted amino acid sequence of the ZAP-70protein. Polyclonal antibodies can be generated by injecting suchpeptides into, for instance, rabbits.

D. Antibodies Raised by Injection of ZAP-70 Encoding Sequence

Antibodies may be raised against the ZAP-70 protein by subcutaneousinjection of a recombinant DNA vector that expresses the ZAP-70 proteininto laboratory animals, such as mice. Delivery of the recombinantvector into the animals may be achieved using a hand-held form of theBiolistic system (Sanford et al., Particulate Sci. Technol. 5:27-37,1987), as described by Tang et al. (Nature 356:152-154, 1992).Expression vectors suitable for this purpose may include those thatexpress the ZAP-70 encoding sequence under the transcriptional controlof either the human β-actin promoter or the cytomegalovirus (CMV)promoter.

Antibody preparations such as those prepared according to theseprotocols are useful in quantitative immunoassays which determineconcentrations of antigen-bearing substances in biological samples; theyare also used semi-quantitatively or qualitatively to identify thepresence of antigen in a biological sample. Alternatively, commerciallyavailable antibodies directed against ZAP-70, such as those listed inTable 1 above, may be used in quantitative and qualitative immunoassaysto identify the presence of the antigen in a biological sample.

Example 6 Nucleic Acid-Based Diagnosis/Detection/Discrimination

The Ig-unmutated CLL-related nucleic acid molecules provided herein, andcombinations of these molecules, can be used in methods of genetictesting for diagnosing, detecting, and/or discriminating between CLL/SLLclinical subgroups or prognosis owing to expression abnormalities in thenucleic acid molecule(s) (e.g., over- or under-expression in comparisonto a control or baseline). For such procedures, a biological sample ofthe subject, which biological sample contains either DNA or RNA derivedfrom the subject, is assayed for over- or under-expression of anIg-unmutated CLL-related nucleic acid molecule. Regulatory regions of agene encoding an Ig-unmutated CLL-related nucleic acid molecule, such asthe enhancer or promoter regions, may also be assayed for theirinvolvement in the over- or under-expression of an Ig-unmutatedCLL-related nucleic acid molecule. Suitable biological samples includesamples containing genomic DNA or RNA (including mRNA), obtained fromcells of a subject, such as those present in peripheral blood, bonemarrow, urine, saliva, tissue biopsy, surgical specimen, amniocentesissamples and autopsy material. Biological samples can be obtained fromnormal, healthy subjects or from subjects who are predisposed to or whoare suffering from a leukemia such as, but not limited to, CLL.

The detection in the biological sample of over- or under-expression ofone or more Ig-unmutated CLL-related nucleic acid molecule(s), may beperformed by a number of methodologies, examples of which are provided.

Over- or under-expression of an Ig-unmutated CLL-related molecule can bedetected by measuring the cellular level of Ig-unmutated CLL-relatednucleic acid molecule-specific mRNA. mRNA can be measured usingtechniques well known in the art, including for instance Microarrayanalysis, Northern analysis, RT-PCR and mRNA in situ hybridization.Details of representative mRNA analysis procedures can be found, forinstance, in Example 1 and Sambrook et al. (ed.), Molecular Cloning: ALaboratory Manual, 2nd ed., vol. 1-0.3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989. Details of representativemicroarray analysis procedures can be found in Example 1, above.

Oligonucleotides used in the above procedures can be labeledradioactively with isotopes (such as ³²P) or non-radioactively, withtags such as biotin or fluorescent dyes (Ward and Langer, Proc. Natl.Acad. Sci. USA 78:6633-6657, 1981), and hybridized to individual DNAsamples immobilized on membranes or other solid supports, for example bydot-blot or transfer from gels after electrophoresis. Quantitative orsemi-quantitative PCR can also be used to measure the amount of anIg-unmutated CLL-related molecule cDNA in a sample using Ig-unmutatedCLL-related molecule oligonucleotide primers. Visualization methods suchas autoradiography or fluorometric (Landegren et al., Science242:229-237, 1989) or colorimetric reactions (Gebeyehu et al., NucleicAcids Res. 15:4513-4534, 1987) can be used to detect a signal and thesignals quantitated using, for instance, a spectrophotometer, ascintillation counter, a densitometer or a Phosphorimager (AmershamBiosciences). The Phosphorimager is able to analyze both DNA and proteinsamples from blots and gels using autoradiographic, direct fluorescenceor chemifluorescence detection. Since the Phosphorimager is moresensitive than ordinary x-ray film, exposure times can be reduced up toten-fold and signal quantitation of both weak and strong signals on thesame blot is possible. Images can be visualized and evaluated with theaid of computer programs such as ImageQuant™.

The nucleic acid-based diagnostic methods of this disclosure arepredictive of CLL disease status, severity, or category. Cells of anysamples that demonstrate abnormal levels (e.g., through over- orunder-expression) of nucleotide sequences that share homology with theIg-unmutated CLL-related nucleic acids disclosed herein are aggressivetumor cells, and result in decreased survival, increased metastasis, andoverall worsened prognosis.

Example 7 ZAP-70 Protein

Another method of discriminating between clinical subgroups of CLL/SLLis to examine, and in some instances quantitate (either comparatively orin absolute terms), the level of ZAP-70 protein in the cells of asubject. This diagnostic tool would be useful for detecting increasedlevels of the ZAP-70 protein that result from, for example, mutations inthe promoter regions of the ZAP-70 gene or mutations within the codingregion of the gene. Alternatively, duplications of the ZAP-70 gene maybe detected as an increase in the expression level of this protein. Thedetermination of increased ZAP-70 protein levels can be used inconjunction with the determination of ZAP-70 mRNA expression levels bythe methods outlined above.

The availability of antibodies specific to the ZAP-70 protein willfacilitate the examination of cellular ZAP-70 protein by one of a numberof immunoassay methods, which are well known in the art and arepresented herein and in, for instance, Harlow and Lane (Antibodies, ALaboratory Manual, CSHL, New York, 1988).

For the purposes of examining the ZAP-70 protein, a biological sample ofthe subject is used, which sample includes cellular proteins. Such abiological sample may be obtained from body cells, such as those presentin peripheral blood, urine, saliva, tissue biopsy, amniocentesissamples, surgical specimens and autopsy material. Biological samples canbe obtained from normal, healthy subjects or from subjects who arepredisposed to or who are suffering from a leukemia such as, but notlimited to, CLL.

Antibodies can be used to assess the presence or absence of ZAP-70 incultured cells or primary cells. The determination that an antibodyspecifically detects the ZAP-70 protein is made by any one of a numberof standard immunoassay methods; for instance, the Western blottingtechnique (Sambrook et al., In Molecular Cloning: A Laboratory Manual,CSHL, New York, 1989). In one embodiment, it is determined whether agiven antibody preparation (such as one produced in a mouse)specifically detects the ZAP-70 protein by Western blotting. In onespecific, non-limiting embodiment total cellular protein is extractedfrom human cells (for example, lymphocytes) and electrophoresed on asodium dodecyl sulfate-polyacrylamide gel. In another embodiment, thecellular protein is extracted from a leukemic cell. The proteins arethen transferred to a membrane (for example, nitrocellulose or PVDF) byWestern blotting, and the antibody preparation is incubated with themembrane. After washing the membrane to remove non-specifically boundantibodies, the presence of specifically bound antibodies is detected bythe use of (by way of example) an anti-mouse antibody conjugated to anenzyme such as alkaline phosphatase. Application of an alkalinephosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro bluetetrazolium results in the production of a dense blue compound byimmunolocalized alkaline phosphatase. Antibodies that specificallydetect the ZAP-70 protein will, by this technique, be shown to bind tothe ZAP-70 protein band (which will be localized at a given position onthe gel determined by its molecular weight, which is approximately 70kDa based on its deduced amino acid sequence). Non-specific binding ofthe antibody to other proteins may occur and may be detectable as a weaksignal on the Western blot. The non-specific nature of this binding willbe recognized by one skilled in the art by the weak signal obtained onthe Western blot relative to the strong primary signal arising from thespecific antibody-ZAP-70 protein binding.

An alternative method of diagnosing ZAP-70 gene deletion, amplification,or a mutation in ZAP-70 regulatory sequences, for example the ZAP-70promoter, is to quantitate the level of ZAP-70 protein in the cells of asubject. In one embodiment, this diagnostic tool would be useful fordetecting increased levels of the ZAP-70 protein that result from, forexample, mutations in the promoter regions of the ZAP-70 gene. Inanother embodiment, duplications of the ZAP-70 gene may be detected asan increase in the expression level of this protein. The determinationof increased ZAP-70 protein levels would be an alternative orsupplemental approach to the direct determination of ZAP-70 geneduplication or mutation status by the methods outlined above.

The availability of antibodies specific to the ZAP-70 protein willfacilitate the quantitation of cellular ZAP-70 protein by one of anumber of immunoassay methods, which are well known in the art and arepresented herein and in, for instance, Harlow and Lane (Antibodies, ALaboratory Manual, CSHL, New York, 1988). Many techniques are commonlyknown in the art for the detection and quantification of antigen. In onespecific, non-limiting embodiment, the purified antigen will be bound toa substrate, the antibody of the sample will bind via its Fab portion tothis antigen, the substrate will then be washed and a second, labeledantibody will then be added which will bind to the Fc portion of theantibody that is the subject of the assay. The second, labeled antibodywill be species specific, i.e., if the serum is from a rabbit, thesecond, labeled antibody will be anti-rabbit-IgG antibody. The specimenwill then be washed and the amount of the second, labeled antibody thathas been bound will be detected and quantified by standard methods.

Examples of methods for the detection of antibodies in biologicalsamples, including methods employing dip strips or other immobilizedassay devices, are disclosed for instance in the following patents: U.S.Pat. Nos. 5,965,356 (Herpes simplex virus type specific seroassay);6,114,179 (Method and test kit for detection of antigens and/orantibodies); 6,077,681 (Diagnosis of motor neuropathy by detection ofantibodies); 6,057,097 (Marker for pathologies comprising an auto-immunereaction and/or for inflammatory diseases); and 5,552,285 (Immunoassaymethods, compositions and kits for antibodies to oxidized DNA bases).

In one embodiment, for the purposes of quantitating the ZAP-70 protein,a biological sample of the subject, as described above and whichincludes cellular proteins, is used. Quantitation of ZAP-70 protein canbe achieved by immunoassay (for example, by ELISA),immunohistochemistry, immunofluorescence, or flow cytometry and comparedto levels of the protein found in healthy cells (e.g., cells from asubject known not to suffer from CLL) followed by spectrophometry ordensitometry. In one embodiment, a significant (e.g., 10% or greater,for instance, 20%, 25%, 30%, 50% or more) increase in the amount ofZAP-70 protein in the cells of a subject compared to the amount ofZAP-70 protein found in normal human cells would be taken as anindication that that a duplication or enhancing mutation had occurred.In this instance, the subject may have Ig-unmutated CLL, and clinicallysevere or progressive CLL.

Example 8 Suppression of ZAP-70 Expression

A reduction of ZAP-70 protein expression in a target cell may beobtained by introducing into cells an antisense or other suppressiveconstruct based on the ZAP-70 encoding sequence, including the humanZAP-70 cDNA (SEQ ID NO: 1) or gene sequence or flanking regions thereof.For antisense suppression, a nucleotide sequence from a ZAP-70 encodingsequence, e.g. all or a portion of the ZAP-70 cDNA or gene, is arrangedin reverse orientation relative to the promoter sequence in thetransformation vector. Other aspects of the vector may be chosen asdiscussed above (Example 4).

The introduced sequence need not be the full length human ZAP-70 cDNA(SEQ ID NO: 1) or gene, and need not be exactly homologous to theequivalent sequence found in the cell type to be transformed. Thus,portions or fragments of the human cDNA (SEQ ID NO: 1) could also beused to knock out or suppress expression of the human ZAP-70 gene.Generally, however, where the introduced sequence is of shorter length,a higher degree of identity to the native ZAP-70 sequence will be neededfor effective antisense suppression. The introduced antisense sequencein the vector may be at least 15 nucleotides in length, and improvedantisense suppression typically will be observed as the length of theantisense sequence increases. The length of the antisense sequence inthe vector advantageously may be greater than 100 nucleotides, and canbe up to about the full length of the human ZAP-70 cDNA or gene. Forsuppression of the ZAP-70 gene itself, transcription of an antisenseconstruct results in the production of RNA molecules that are thereverse complement of mRNA molecules transcribed from the endogenousZAP-70 gene in the cell.

Although the exact mechanism by which antisense RNA molecules interferewith gene expression has not been elucidated, it is believed thatantisense RNA molecules bind to the endogenous mRNA molecules andthereby inhibit translation of the endogenous mRNA. Expression of ZAP-70can also be reduced using small inhibitory RNAs, for instance usingtechniques similar to those described previously (see, e.g., Tuschl etal., Genes Dev 13, 3191-3197, 1999; Caplen et al., Proc. Nat.l Acad.Sci. U.S.A. 98, 9742-9747, 2001; and Elbashir et al., Nature 411,494-498, 2001).

Suppression of endogenous ZAP-70 expression can also be achieved usingribozymes. Ribozymes are synthetic RNA molecules that possess highlyspecific endoribonuclease activity. The production and use of ribozymesare disclosed in U.S. Pat. No. 4,987,071 to Cech and U.S. Pat. No.5,543,508 to Haselhoff. The inclusion of ribozyme sequences withinantisense RNAs may be used to confer RNA cleaving activity on theantisense RNA, such that endogenous mRNA molecules that bind to theantisense RNA are cleaved, which in turn leads to an enhanced antisenseinhibition of endogenous gene expression.

Inhibition of ZAP-70 can be achieved by using agents, such as drugs,that target the protein itself. Examples of agents that could inhibitZAP-70 function include kinase inhibitors and molecular decoys (drugsthat affect protein-protein interactions). Dominant negative mutantforms of ZAP-70 may also be used to block endogenous ZAP-70 activity.

Example 9 ZAP-70 Knockout and Overexpression Transgenic Animals

Mutant organisms that under-express or over-express ZAP-70 protein areuseful for research, for instance for testing and analyzing putativepharmaceutical agents useful in controlling ZAP-70 expression oractivity. Such mutants allow insight into the physiological and/orpathological role of ZAP-70 in a healthy and/or pathological organism.These mutants are “genetically engineered,” meaning that information inthe form of nucleotides has been transferred into the mutant's genome ata location, or in a combination, in which it would not normally exist.Nucleotides transferred in this way are said to be “non-native.” Forexample, a non-ZAP-70 promoter inserted upstream of a native ZAP-70 genewould be non-native. An extra copy of a ZAP-70 gene or other encodingsequence on a plasmid, transformed into a cell, would be non-native,whether that extra copy was ZAP-70 derived from the same, or a differentspecies.

Mutants may be, for example, produced from mammals, such as mice, thateither over-express or under-express ZAP-70 protein, or that do notexpress ZAP-70 at all. Over-expression mutants are made by increasingthe number of ZAP-70-encoding sequences (such as genes) in the organism,or by introducing an ZAP-70-encoding sequence into the organism underthe control of a constitutive or inducible or viral promoter such as themouse mammary tumor virus (MMTV) promoter or the whey acidic protein(WAP) promoter or the metallothionein promoter. Mutants thatunder-express ZAP-70 may be made by using an inducible or repressiblepromoter, by deleting the ZAP-70 gene, by destroying or limiting thefunction of the ZAP-70 gene, for instance by disrupting the gene bytransposon insertion, or by RNA interference (RNAi).

Antisense genes may be engineered into the organism, under aconstitutive or inducible promoter, to decrease or prevent ZAP-70expression, as discussed above in Example 15.

A gene is “functionally deleted” when genetic engineering has been usedto negate or reduce gene expression to negligible levels. When a mutantis referred to in this application as having the ZAP-70 gene altered orfunctionally deleted, this refers to the ZAP-70 gene and to any orthologof this gene. When a mutant is referred to as having “more than thenormal copy number” of a gene, this means that it has more than theusual number of genes found in the wild-type organism, e.g., in thediploid mouse or human.

In Caenorhabditis elegans, double stranded RNA (dsRNA) mediated genesilencing, RNAi (Fire et al., Nature 391, 806-811, 1998) has beenapplied to generate “somatic knockouts” for the functional analysis ofgenes (Fraser et al., Nature 408, 325-330, 2000; Gonczy et al., Nature408, 331-336, 2000). In mammalian cells, it has recently beendemonstrated that synthetic 20-23 nucleotide (nt) dsRNA molecules orsmall interfering RNAs (siRNAs) (Tuschl et al., Genes Dev 13, 3191-3197,1999) can induce RNAi gene silencing without activation of non-specificdsRNA-dependent pathways (Caplen et al., Proc. Nat.l Acad. Sci. U.S.A.98, 9742-9747, 2001; Elbashir et al., Nature 411, 494-498, 2001).

Several models have been put forward to explain RNAi, in particular themechanisms by which the cleavage derived small dsRNAs or siRNAs interactwith the target mRNA and thus facilitate its degradation (Hamilton etal., Science 286, 950, 1999; Zamore et al., Cell 101, 25, 2000; Hammondet al., Nature 404, 293, 2000; Yang et al., Curr. Biol. 10, 1191, 2000;Elbashir et al., Genes Dev. 15, 188, 2001; Bass Cell 101, 235, 2000). Ithas been proposed that the cleavage derived small dsRNAs or siRNAs actas a guide for the enzymatic complex required for the sequence specificcleavage of the target mRNA. Evidence for this includes cleavage of thetarget mRNA at regular intervals of ˜21-23 nts in the regioncorresponding to the input dsRNA (Zamore et al., Cell 101, 25, 2000),with the exact cleavage sites corresponding to the middle of sequencescovered by individual 21- or 22 nt small dsRNAS or siRNAs (Elbashir etal., Genes Dev. 15, 188, 2001). Although mammals and lower organismsappear to share dsRNA-triggered responses that involve a relatedintermediate (small dsRNAs), it is likely that there will be differencesas well as similarities in the underlying mechanism.

ydsRNAs can be formed from RNA oligomers produced synthetically (fortechnical details see material from Xeragon and Dharmacon, bothavailable on the internet). Small dsRNAs and siRNAs can also bemanufactured using standard methods of in vitro RNA production. See, forinstance, methods and characteristics described in U.S. ProvisionalPatent Application No. 60/308,640 (filed Jul. 30, 2001, and incorporatedherein by reference). In addition, the Silencer™ siRNA Construction kit(and components thereof) available from Ambion (Catalog #1620; Austin,Tex.), which employs a T7 promoter and other well known geneticengineering techniques to produce dsRNAs. Double stranded RNA triggerscould also be expressed from DNA based vector systems.

A mutant mouse over-expressing ZAP-70 may be made by constructing aplasmid having the ZAP-70 gene driven by a promoter, such as the mousemammary tumor virus (MMTV) promoter or the whey acidic protein (WAP)promoter. This plasmid may be introduced into mouse oocytes bymicroinjection. The oocytes are implanted into pseudopregnant females,and the litters are assayed for insertion of the transgene. Multiplestrains containing the transgene are then available for study.

WAP is quite specific for mammary gland expression during lactation, andMMTV is expressed in a variety of tissues including mammary gland,salivary gland and lymphoid tissues. Many other promoters might be usedto achieve various patterns of expression, e.g., the metallothioneinpromoter.

An inducible system may be created in which the subject expressionconstruct is driven by a promoter regulated by an agent that can be fedto the mouse, such as tetracycline. Such techniques are well known inthe art.

A mutant knockout animal (e.g., mouse) from which the ZAP-70 gene isdeleted or otherwise disabled can be made by removing coding regions ofthe ZAP-70 gene from embryonic stem cells. The methods of creatingdeletion mutations by using a targeting vector have been described (see,for instance, Thomas and Capecch, Cell 51:503-512, 1987).

Example 10 Nucleic Acid-Based ZAP-70 Therapy

Gene therapy approaches for combating ZAP-70-mediated defects insubjects, such as uncontrolled or disregulated cell growth or neoplasm,are now made possible.

Retroviruses have been considered a preferred vector for experiments ingene therapy, with a high efficiency of infection and stable integrationand expression (Orkin et al., Prog. Med. Genet. 7:130-142, 1988). Thefull-length ZAP-70 gene or cDNA can be cloned into a retroviral vectorand driven from either its endogenous promoter or, for instance, fromthe retroviral LTR (long terminal repeat). Other viral transfectionsystems may also be utilized for this type of approach, includingadenovirus, adeno-associated virus (AAV) (McLaughlin et al., J. Virol.62:1963-1973, 1988), Vaccinia virus (Moss et al., Annu. Rev. Immunol.5:305-324, 1987), Bovine Papilloma virus (Rasmussen et al., MethodsEnzymol. 139:642-654, 1987) or members of the herpesvirus group such asEpstein-Barr virus (Margolskee et al., Mol. Cell. Biol. 8:2837-2847,1988).

More recent developments in gene therapy techniques include the use ofRNA-DNA hybrid oligonucleotides, as described by Cole-Strauss, et al.(Science 273:1386-1389, 1996). This technique may allow forsite-specific integration of cloned sequences, thereby permittingaccurately targeted gene replacement.

In addition to delivery of ZAP-70 to cells using viral vectors, it ispossible to use non-infectious methods of delivery. For instance,lipidic and liposome-mediated gene delivery has recently been usedsuccessfully for transfection with various genes (for reviews, seeTempleton and Lasic, Mol. Biotechnol. 11:175-180, 1999; Lee and Huang,Crit. Rev. Ther. Drug Carrier Syst. 14:173-206; and Cooper, Semin.Oncol. 23:172-187, 1996). For instance, cationic liposomes have beenanalyzed for their ability to transfect monocytic leukemia cells, andshown to be a viable alternative to using viral vectors (de Lima et al.,Mol. Membr. Biol. 16:103-109, 1999). Such cationic liposomes can also betargeted to specific cells through the inclusion of, for instance,monoclonal antibodies or other appropriate targeting ligands (Kao etal., Cancer Gene Ther. 3:250-256, 1996).

Example 11 Kits

Kits are provided which contain the necessary reagents for determiningabnormal expression of ZAP-70 mRNA or ZAP-70 protein. Instructionsprovided in the diagnostic kits can include calibration curves,diagrams, illustrations, or charts or the like to compare with thedetermined (e.g., experimentally measured) values or other results.

A. Kits for Detection of ZAP-70 mRNA Expression

Kits similar to those disclosed above for the detection of ZAP-70genomic sequences can be used to detect ZAP-70 mRNA expression levels.Such kits may include an appropriate amount of one or more of theoligonucleotide primers for use in reverse transcription amplificationreactions, similarly to those provided above, with art-obviousmodifications for use with RNA.

In some embodiments, kits for detection of ZAP-70 mRNA expression levelsmay also include the reagents necessary to carry out RT-PCR in vitroamplification reactions, including, for instance, RNA sample preparationreagents (including e.g., an RNAse inhibitor), appropriate buffers(e.g., polymerase buffer), salts (e.g., magnesium chloride), anddeoxyribonucleotides (dNTPs). Written instructions may also be included.

Kits in addition may include either labeled or unlabeled oligonucleotideprobes for use in detection of the in vitro amplified target sequences.The appropriate sequences for such a probe will be any sequence thatfalls between the annealing sites of the two provided oligonucleotideprimers, such that the sequence the probe is complementary to isamplified during the PCR reaction.

It also may be advantageous to provided in the kit one or more controlsequences for use in the RT-PCR reactions. The design of appropriatepositive control sequences is well known to one of ordinary skill in theappropriate art.

The kit may also include the necessary reagents to perform thepurification of the clinical sample or the normalization of the amountof cells present in the sample.

Alternatively, kits may be provided with the necessary reagents to carryout quantitative or semi-quantitative Northern analysis of ZAP-70 mRNA.Such kits include, for instance, at least one ZAP-70-specificoligonucleotide for use as a probe. This oligonucleotide may be labeledin any conventional way, including with a selected radioactive isotope,enzyme substrate, co-factor, ligand, chemiluminescent or fluorescentagent, hapten, or enzyme.

B. Kits For Detection of ZAP-70 Protein or Peptide Expression

Kits for the detection of ZAP-70 protein expression, include forinstance at least one target protein specific binding agent (e.g., apolyclonal or monoclonal antibody or antibody fragment) and may includeat least one control. The ZAP-70 protein specific binding agent andcontrol may be contained in separate containers. The kits may alsoinclude means for detecting ZAP-70:agent complexes, for instance theagent may be detectably labeled. If the detectable agent is not labeled,it may be detected by second antibodies or protein A for example whichmay also be provided in some kits in one or more separate containers.Such techniques are well known.

The kit may also include the necessary reagents to perform thepurification of the clinical sample or the normalization of the amountof cells present in the sample.

Additional components in some kits include instructions for carrying outthe assay. Instructions will allow the tester to determine whetherZAP-70 expression levels are altered, for instance in comparison to acontrol sample. Reaction vessels and auxiliary reagents such aschromogens, buffers, enzymes, etc. may also be included in the kits.

By way of example only, an effective and convenient immunoassay kit suchas an enzyme-linked immunosorbent assay can be constructed to testanti-ZAP-70 antibody in human serum. Expression vectors can beconstructed using the human ZAP-70 cDNA to produce the recombinant humanZAP-70 protein in either bacteria or baculovirus (as described inExample 10). By affinity purification, unlimited amounts of purerecombinant ZAP-70 protein can be produced.

An assay kit could provide the recombinant protein as an antigen andenzyme-conjugated goat anti-human IgG as a second antibody as well asthe enzymatic substrates. Such kits can be used to test if the patientsera contain antibodies against human ZAP-70.

This disclosure provides methods of determining or detecting diseasestatus in a subject, particularly detecting, determining, ordiscriminating between clinical subgroups of CLL/SLL based on the levelsof mRNA or protein levels in a biological sample from a subject. Thedisclosure further provides compositions for use in such methods,pharmaceutical preparations, and kits and assays. It will be apparentthat the precise details of the methods and compositions described maybe varied or modified without departing from the spirit of the describedinvention. We claim all such modifications and variations that fallwithin the scope and spirit of the claims below.

1. A method of determining a predisposition to poor clinical outcome ina subject having chronic lymphocytic leukemia (CLL), comprisingdetermining in a sample comprising B-cells from the subject whether oneor more of the B-cells are ZAP-70 positive, wherein ZAP-70 positiveB-cells indicate predisposition to poor clinical outcome.
 2. The methodof claim 1, wherein the B-cells comprise CLL cells.
 3. The method ofclaim 1, wherein the predisposition to poor clinical outcome iscorrelated with the presence of Ig-unmutated CLL in the subject withCLL.
 4. The method of claim 1, wherein determining whether one or moreof the B-cells are ZAP-70 positive comprises: contacting the sample fromthe subject with a reagent comprising a ZAP-70-specific antibody orbinding fragment thereof, to form a ZAP-70:antibody complex or aZAP-70:antibody binding fragment complex; and detecting the complex. 5.The method of claim 4, wherein detecting the complex comprises flowcytometry, immunocytochemistry, Western blot, or ELISA.
 6. The method ofclaim 1, wherein the ZAP-70 comprises a polypeptide comprising the aminoacid sequence of SEQ ID NO:
 2. 7. The method of claim 1, furthercomprising determining whether one or more of the B-cells overexpressesa second polypeptide.
 8. The method of claim 7, wherein the secondpolypeptide is an activation-induced C-type lectin polypeptide.
 9. Themethod of claim 7, further comprising determining whether one or more ofthe B-cells overexpresses a third polypeptide.
 10. A method ofdetermining a predisposition to poor clinical outcome in a subjecthaving chronic lymphocytic leukemia (CLL), comprising determining in asample comprising B-cells from the subject whether one or more of theB-cells expresses a ZAP-70 polypeptide, wherein expression of the ZAP-70polypeptide indicates predisposition to poor clinical outcome.
 11. Themethod of claim 10, wherein the B-cells comprise CLL cells.
 12. Themethod of claim 10, wherein the predisposition to poor clinical outcomeis correlated with the presence of Ig-unmutated CLL in the subject withCLL.
 13. The method of claim 10, wherein determining whether one or moreof the B-cells expresses the ZAP-70 polypeptide comprises: contactingthe sample from the subject with a reagent comprising a ZAP-70-specificantibody or binding fragment thereof, to form a ZAP-70:antibody complexor a ZAP-70:antibody binding fragment complex; and detecting thecomplex.
 14. The method of claim 13, wherein detecting the complexcomprises flow cytometry, immunocytochemistry, Western blot, or ELISA.15. The method of claim 10, wherein the ZAP-70 polypeptide comprises apolypeptide comprising the amino acid sequence of SEQ ID NO:
 2. 16. Themethod of claim 10, further comprising determining whether one or moreof the B-cells overexpresses a second polypeptide.
 17. The method ofclaim 16, wherein the second polypeptide is an activation-induced C-typelectin polypeptide.
 18. The method of claim 16, further comprisingdetermining whether one or more of the B-cells overexpresses a thirdpolypeptide.
 19. A method of determining a predisposition to poorclinical outcome in a subject having chronic lymphocytic leukemia (CLL),comprising determining in a sample comprising B-cells from the subjectwhether one or more of the B-cells are ZAP-70 positive, wherein theZAP-70 comprises a ZAP-70 polypeptide of SEQ ID NO: 2, and whereinZAP-70 positive B-cells indicates predisposition to poor clinicaloutcome.
 20. The method of claim 19, wherein determining whether one ormore of the B-cells are ZAP-70 positive comprises: reacting at least oneZAP-70 polypeptide of SEQ ID NO: 2 contained in the sample with areagent comprising a ZAP-70-specific antibody to form a ZAP-70:antibodycomplex; and detecting the complex using flow cytometry, whereinpresence of the complex indicates the one or more B-cells are ZAP-70positive.
 21. A method of determining a predisposition to poor clinicaloutcome in a subject having chronic lymphocytic leukemia (CLL),comprising determining in a sample comprising B-cells from the subjectwhether one or more of the B-cells express a ZAP-70 polypeptide of SEQID NO: 2, wherein expression of the ZAP-70 polypeptide indicatespredisposition to poor clinical outcome.
 22. The method of claim 21,wherein determining whether one or more of the B-cells expresses theZAP-70 polypeptide comprises: reacting at least one ZAP-70 polypeptideof SEQ ID NO: 2 contained in the sample with a reagent comprising aZAP-70-specific antibody to form a ZAP-70:antibody complex; anddetecting the complex using flow cytometry.